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Department of Pharmacology University of Nebraska Medical Center Omaha, Nebraska 68198-6260
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hsp90 and p23 play important roles in establishing and stabilizing the high-affinity hormone-binding conformation of GR and PR, but the functional requirement for immunophilins in mature receptor complexes has been difficult to define. Perhaps the best in vivo evidence of a role for Hsp90-associated immunophilins in steroid receptor function comes from a heterologous model in which a vertebrate steroid receptor is expressed in Saccharomyces cerevisiae. In the S. cerevisiae genomic sequence, there is no clear homolog for the large FKBPs, but there are two Cyp40 homologs, termed Cpr6 and Cpr7 (4). Both Cpr6 (5) and Cpr7 (6) bind to Hsp90, but only Cpr7 genetically interacts with GR. Deletion of the yeast gene for Cpr7, but not the Cpr6 gene, caused an 80% reduction in GR-dependent activation of a reporter gene (6).
FKBP52, FKBP51 and Cyp40 have peptidylprolyl isomerase (PPIase) activity, although the functional importance of this activity is an open question. The immunosuppressant FK506 binds the PPIase active site and blocks enzymatic activity, but FK506 has no apparent effect on the composition of PR complexes (7) or on the composition and in vitro assembly of GR complexes (8). FKBP52 in GR complexes will bind an FK506 affinity resin without dissociating from the receptor complex (9), but FKBP51 binds well to an FK506 resin only when it is dissociated from Hsp90 and other proteins (10).
A number of cellular studies examining the effects of immunosuppressant drugs on steroid hormone action have reported potentiation or attenuation of steroid responses (11, 12, 13, 14). These findings have been interpreted to support a role for receptor-associated immunophilins in normal receptor function; however, there are multiple immunophilin and nonimmunophilin target sites for these drugs in cells, and it has not been demonstrated that receptor-associated immunophilins are directly responsible for the reported pharmacological effects. Pratt and colleagues (15, 16) have succeeded in assembling GR into functionally mature complexes in a reconstituted system that lacks immunophilins, arguing against a role for immunophilins in hormone binding. The Pratt group (17) has proposed that FKBP52 may participate in the nuclear translocation of GR complexes. Evidence was presented that FKBP52 may bind a nuclear localization sequence (NLS) of GR, thus serving as an NLS receptor (18, 19), but this purported function for FKBP52 has not been conclusively demonstrated.
Human FKBP51 shares 60% amino acid sequence identity and 75% similarity with human FKBP52, and both are expressed constitutively in many mammalian cell and tissue types. FKPB51 is up-regulated during adipogenesis (20) and in response to glucocorticoids (21), and expression of FKBP52 is stimulated by several mitogenic growth factors (22). We have observed a dexamethasone-dependent increase of FKBP51 protein levels, but not FKBP52 levels, in rat L929 fibroblasts and HTC19.G11 cells (2-fold and 5-fold increases, respectively; Y. Ruan and D. F. Smith, unpublished). A potentially relevant correlation between FKBP expression levels and GR activity has recently been made. In B-lymphoblast extracts from glucocorticoid-resistant squirrel monkeys, where a cytosolic factor appears to be responsible for GR’s lower hormone-binding affinity (23), FKBP51 is overexpressed and FKBP52 is greatly underexpressed relative to type-matched human cells (24).
The best characterized interactions of FKBP52 and FKBP51 are with Hsp90 and steroid receptor complexes. In rabbit reticulocyte lysate (RL), a medium used for cell-free assembly studies of steroid receptor complexes, FKBP51 is present at approximately 20 nM as compared with 100, 200, and 1,000 nM concentrations for, respectively, FKBP52, Cyp40, and Hsp90 (25). Despite its lower concentration, FKBP51 was preferentially recovered in PR complexes (25), suggesting a potential functional difference in FKBP51 and FKBP52. Previous observations have also distinguished the behaviors of FKBP51 and FKBP52. In native chicken PR complexes, FKBP51’s association is uniquely sensitive to hormone binding and to sulfhydryl-modifying agents (26, 27). Unlike FKBP52, FKBP51 in heteromeric complexes binds poorly to an FK506 affinity matrix, suggesting that FKBP51’s PPIase domain is less accessible. In the present report, we have more closely examined FKBP51’s preferential association with steroid receptor complexes and have mapped out the region of FKBP51 responsible for its preference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), it differed from an earlier analysis
(2) by having a greater ratio of RL to PR in the assembly mixture.
Recombinant PR-A was bound to an immunoaffinity resin and placed in RL
supplemented with radiolabeled chaperone components. Aliquots were
removed from the assembly mixture over a 60-min time course and
immediately quenched in cold wash buffer. Resin complexes were
separated by SDS-PAGE, and the gels were Coomassie-stained for total
protein levels and autoradiographed to visualize radiolabeled proteins.
The absorbance values for bands on x-ray film were normalized to the
amount of Coomassie-stained receptor in each lane, and the values were
plotted as a percentage of the maximum recovery for each component.
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, the recoveries of
Hsp70, Hip, and Hop with PR complexes all attain a maximum in the first
5–10 min and then drop to lower levels. Hsp90 recovery contrasts by
reaching a sustained plateau of maximal binding in 10–15 min. These
patterns are consistent with an earlier analysis (2). Expansion of the
scale to better resolve changes over the first 5 min (inset)
reveals that Hsp70 binding precedes the binding of Hip and Hop,
reflecting the initial formation of an early PR complex with Hsp70,
followed by intermediate complexes containing Hip, Hop, and Hsp90. The
delayed plateau of Hsp90 binding reflects accumulation of relatively
stable mature complexes, which lack Hsp70, Hip, and Hop, as assembly
reaches steady state conditions.
Immunophilins, which are restricted to mature PR complexes, display two
patterns of recovery with PR complexes (Fig. 1, lower
panel). Cyp40 and FKBP52 achieve maximal binding levels over 5–15
min but decrease thereafter. In contrast, FKBP51 levels do not reach a
maximum until 30 min after initiation of assembly. These changes are
consistent with FKBP51 being competitively preferred in mature PR
complexes but also being present at a lower concentration in RL than
FKBP52 and Cyp40.
It was shown qualitatively that ER complexes assembled in
vitro contain FKBP51 (3), but FKBP51’s binding was not
quantitated relative to FKBP52 and Cyp40. To our knowledge, GR
complexes have not been directly tested for FKBP51. In Fig. 2, the immunophilin compositions of
in vitro-assembled PR, GR, and ER complexes were compared.
In preliminary characterizations, immunoprecipitation levels for each
of the three receptors were quantitated by densitometry of
Coomassie-stained receptor bands on one-dimensional SDS gels (not
shown). For compositional determinations, approximately 0.5 µg of
receptor was incubated with 500 µl RL under maximal assembly
conditions for 45 min. PR complexes contained the typical proportions
of immunophilins with FKBP51 > Cyp40 > FKBP52.
Surprisingly, GR complexes contained an even higher proportion of
FKBP51, apparently at the expense of Cyp40. ER complexes had relatively
higher proportions of Cyp40 and FKBP52 than observed in PR and GR
complexes. The relative association levels seen in Fig. 2 were
consistently observed in replicate receptor assemblies.
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). The PPIase domain indicated on the
alignment (single underline) corresponds to the region of
homology to FKBP12. The tetratricopeptide repeat (TPR) region contains
three TPR units (double underlines), and there is a
consensus calmodulin-binding motif (CaMBC; dashed underline)
downstream from the TPRs. The putative NLS recognition sequence of
FKBP52 (amino acids 143–147) is indicated by a gray bar.
Interestingly, the corresponding sequence of FKBP51 lacks two of the
glutamic acids and has a phenylalanine in place of FKBP542’s
threonine. No other deletions/insertions are apparent in comparing the
overall sequences.
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, a number of FKBP mutants were prepared. The
small box filled with slanted lines in FKBP51, corresponding
to the putative NLS-binding sequence in FKBP52, indicates the apparent
deletion of two amino acids. In the TPR domain, the first shaded
box corresponds to the first TPR unit, and the larger box
corresponds to the second and third TPR units. FD67DV is a double-point
mutation generated in the PPIase domain of FKBP51 only. A set of
truncation mutants for both FKBP51 and FKBP52 was prepared; only the
FKBP52 mutants are named on the left, but the corresponding
FKBP51 mutants are each shorter by two amino acids. Two sets of
chimeric swap mutants were generated. All were analyzed, but only those
chimeras used in
Figs. 5–7 are named. The first part of the name (52
or 51) indicates the host sequence (FKBP52 or FKBP51, respectively),
the lettered portion designates the particular region swapped, and the
final number (1 or 2) indicates the source for the swapped region
(FKBP51 or FKBP52, respectively).
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Purified, recombinant wild-type FKBP51 (wt51) and mutant FD67DV were
tested in a PPIase assay, and enzymatic activity of the mutant was
reduced by more than 90% relative to wt51 (Fig. 4A). However, radiolabeled wt51 and
FD67DV associated equally with PR complexes during cell-free assembly
(Fig. 4B). Thus, PPIase activity does not appear to be important for
FKBP51 assembly or preferred association with PR complexes.
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, cDNAs
encoding truncated protein products were expressed in vitro
to generate radiolabeled protein products, and these products were
compared for their abilities to coprecipitate with Hsp90 or to
associate with PR complexes. Truncations of the entire sequences
upstream (51/270-C and 52/272-C) or downstream (51/N-389 or 52/N-391)
from the TPR domain were examined first. Equimolar amounts of the
radiolabeled wild-type or mutant proteins (Fig. 5A, gel autoradiograph
in the bottom panel) were added to RL, and the mixtures were
immunoprecipitated with the anti-Hsp90 antibody H90–10. The 51/270-C
lane contains two radiolabeled forms consistent with initiation of
cell-free translation from both Met270 and the internal
Met285. The protein profiles of Hsp90 immunoprecipitates from RL mixtures were visualized on a Coomassie-stained gel (top panel). Note that similar levels of Hsp90 and coprecipitating rabbit proteins were obtained for each sample. Bands representing Hsp90 and the major associated proteins Hop and Hsp70 are identified on the left along with the H90–10 heavy and light chains (HC and LC). The dried gel was autoradiographed to reveal any radiolabeled FKBP forms coprecipitating with Hsp90 (middle panel). Only the full-length FKBPs coprecipitated with Hsp90, suggesting that sequences both upstream and downstream of the TPR domain are necessary for Hsp90 binding. However, the conformational level at which the deleted sequences are needed for TPR binding to Hsp90 is not immediately apparent from these large truncations.
A similar assay was performed for association of FKBP forms with PR; as with Hsp90 complexes, only the full-length FKBPs were recovered in PR complexes (results not shown). The concordance of Hsp90 and PR coprecipitation patterns is not surprising here since the FKBPs, at least initially, indirectly associate with PR through their binding to Hsp90.
The gel and autoradiograph images shown in Fig. 5, B and C, and all
remaining figures were selected to illustrate relative binding levels
of various FKBP mutant forms. Similar to Fig. 5A (bottom
panel), in each set of analyses the specific activity of
radiolabeled mutant forms was determined, and molar amounts of
radiolabeled mutant forms added to assembly mixtures were adjusted as
indicated in the figure legends. Furthermore, all relative binding
results illustrated are consistent with at least two additional
replicate experiments.
FKBP mutants, progressively truncated from the C
terminus toward the TPR domain, were tested for Hsp90 binding and PR
association. FKBP52 binding to Hsp90 (Fig. 5B) was maintained when 43
amino acids were truncated from the C terminus (52/N-416) but not when
an additional 25 amino acids, including the CaMBC, were removed (Fig. 5A, 52/N-391). FKBP51 differs from FKBP52, since Hsp90 binding requires
sequences beyond the CaMBC. Mutant 51/N-414 failed to bind Hsp90,
contrasting with the retention of binding by the corresponding FKBP52
mutant (52/N-416); however, mutant 51/N-431 retains Hsp90 binding.
Qualitatively, association of FKBP truncation mutants with PR (Fig. 5C)
followed a similar pattern as that seen for Hsp90 binding. 52/N-416 was
recovered in PR complexes, although at a lower level than wt52 or
mutants 52/N-433 and 52/N-448 having lesser truncations. Recovery of
FKBP51 mutants was either at levels equivalent to wt 51 (51/N-446 and
51/N-431) or, in agreement with loss of Hsp90 binding, was absent
(51/N-414). The quantitative differences in PR-associated proteins were
consistently observed in two additional assays.
FKBP Chimeras Involving the TPR Domain
The entire TPR domain, including the first TPR unit, a non-TPR
intervening sequence, and the final two TPR units in tandem, was
swapped between FKBP51 and FKBP52 (Fig. 6A). As in Fig. 5A, bottom
panel, radiolabeled wild-type and chimeric constructs were
generated and examined by SDS-PAGE and autoradiography for expression
levels and specific activity (not shown). In all cases, expression
levels were similar. FKBP51 containing the TPR domain from FKBP52
(51T2) displayed preferential association with PR complexes similar to
wt51 (left-hand panel). Unexpectedly, however, the
corresponding FKBP52 chimera containing the TPR from FKBP51 (52T1)
failed to associate with PR, even though this chimera was present in
the mixture at a level equivalent to wt52 or 51T2. Upon examination of
Hsp90 binding by the chimeras (right-hand panel), it was
found that 51T2 bound similar to wild-type FKBPs while 52T1 failed to
bind Hsp90, thus accounting for 52T1’s absence in PR complexes.
To more closely identify sequences within the TPR domain of FKBP51
responsible for loss of Hsp90 binding in chimeric FKBP52, chimeras
between the less highly conserved intervening sequences were tested,
but no changes in Hsp90 binding or PR association were observed
(results not shown). Similarly, the first TPR unit was swapped, but
again no changes in protein interactions were observed. Finally, the
second and third tandem TPR units were swapped, and the effects seen
with complete TPR swaps were replicated (Fig. 5B). Thus, it appears
that some feature localized to the second and third TPR units of FKBP51
requires an additional FKBP51 sequence for Hsp90 binding. FKBP52 TPR
units apparently do not require this additional sequence since they
function equally well in the FKBP52 or FKBP51 background (wt52 and
51Tbc2). FKBP51 chimeras were generated in which a comprehensive set of
FKBP52 sequences upstream or downstream from the TPR were swapped into
FKBP51. These were then tested for coprecipitation with Hsp90. However,
none of these chimeras displayed the loss of Hsp90 binding that would
be expected if a required sequence in FKBP51 had been replaced (results
not shown). Additional studies will be needed to understand the context
dependence of FKBP51’s TPR domain.
FKBP Chimeras Involving the C-Terminal Region
Chimeras swapping FKBP sequences from the end of the TPR domain
down to the C terminus were tested for Hsp90 binding and PR
association. No differences were found between chimeras and wild type
in binding to Hsp90 (results not shown), but quantitative differences
were noted in PR association (Fig. 7A).
The C-terminal 70 amino acids of FKBP51 confer preferred PR association
to FKBP52 (52C1); conversely, the corresponding region of FKBP52
removes preferred association from FKBP51 (51C2).
This region was subdivided to resolve sequences within the C terminus
of FKBP51 responsible for preferred PR association. The exchanged
region in 52V1 (Fig. 7B) lacks the CaMBC but contains the more variable
C-terminal 40 amino acids. Preferred PR association followed this
fragment. This region was further subdivided such that chimera 52Va1
(Fig. 7C) contained only amino acids 404–433 of FKBP51. Preferred PR
association was observed with this chimera.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). The results of an in vitro PR assembly
time course analysis (Fig. 1) suggests that the less abundant
FKBP51-Hsp90 complex has a competitive advantage in PR associations
over more abundant FKBP52- or Cyp40-Hsp90 complexes. To aid in mapping
the sequences in FKBP51 responsible for its preferred association with
PR complexes, a variety of FKBP mutants were generated and tested for
Hsp90 binding and PR association. The results from all tests, those
illustrated in
Figs. 4–7 as well as others, are summarized in Fig. 8A. Two conclusions from this study are
that the TPR domain of FKBP51 is context-sensitive for Hsp90 binding
(Fig. 8B, region 1) and that preferred PR binding maps to a variable
sequence adjacent to the CaMBC of FKBP51 (region 2).
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). The
putative NLS-binding sequence was swapped between FKBP51 and FKBP52,
but no difference was noted in the in vitro assembly of
chimeras with PR (not shown). It is interesting, however, that FKBP51
in comparison with FKBP52 has two less glutamic acid residues and a
threonine to phenylalanine substitution, changes that would reasonably
be expected to alter any NLS-binding activity. Perhaps functional
differences related to this sequence would be noted in the cellular
context, or perhaps this sequence is not an NLS-binding site in either
FKBP.
Since Hsp90 binding is a necessary characteristic for immunophilins to
enter receptor complexes, the TPR/Hsp90-binding domain might be
expected to alter interactions of the immunophilins with steroid
receptors. In one sense this is true, as will be discussed below in
relation to Hsp90 binding, but in the sense of conferring FKBP51’s
preferred association with PR complexes, this is not the case. As shown
in Fig. 6, replacing FKBP51’s TPR segments with those from FKBP52 does
not alter preferred PR association.
Finally, differential interactions of FKBPs with calmodulin through
their CaMBCs, which are similar but nonidentical, might influence PR
associations. Again, the results in Fig. 7 argue against a role for
this sequence in preferred association. In fact, we have reported
earlier (34) and have found through additional unpublished experiments
that the assembly of PR complexes in vitro is no different
when performed in an excess of Ca2+ or in the presence of
the specific Ca2+ chelator EGTA. Some reports (35, 36) have
suggested a role for calmodulin in GR function in vivo, but
the exact mechanism for calmodulin action has not been resolved.
A sequence sufficient for conferring preferred association with PR
complexes maps to the 15–20 amino acid region of FKBP51 immediately
downstream from the CaMBC (Fig. 7). There is little similarity between
FKBP51 and FKBP52 in this region, and it is not clear how this region
influences immunophilin·receptor associations.
Hsp90 Binding by FKBPs
Previous studies on FKBP52 (37) and Cyp40 (38, 39) first
identified the TPR domain of these immunophilins as required for Hsp90
binding. The present studies support the role of FKBP TPR domains in
Hsp90 binding but extend the requirement to sequences outside the TPR.
FKBP truncation mutants lacking sequences either up- or downstream from
the TPR domain failed to bind Hsp90 (Fig. 5A). Upstream sequences
required for Hsp90 binding were not further analyzed, but downstream
sequences were. FKBP52 and FKBP51 differed in that Hsp90 binding was
retained in an FKBP52 truncation ending with the CaMBC site while
FKBP51 binding to Hsp90 was only observed when an additional 20 amino
acids were present (Fig. 5B). Perhaps not coincidentally, these
additional amino acids of FKBP51 colocalize with the sequence that
confers preferential association with PR complexes (Fig. 7C).
A more striking difference in the FKBPs was observed with chimeras
involving the TPR domain (Fig. 6). In FKBP52 chimeras containing the
entire TPR domain or only the second and third repeats of FKBP51, no
Hsp90 binding was observed. Interestingly, though, the converse FKBP51
chimeras retained Hsp90 binding. Whether the behavioral difference in
FKBP51’s TPR resides strictly within its latter TPR units or also
involves extraneous regions of FKBP51 has not been resolved, but the
distinction in the corresponding TPR regions suggest structural
differences in the two FKBPs that could relate to distinctive
interactions with Hsp90. In a recent study comparing immunophilin
binding by an assortment of Hsp90 mutants, several Hsp90 mutants,
either deletions or a point mutation in the C-terminal half of Hsp90,
could distinguish the two FKBPs (29). Thus, while the immunophilins
bind competitively to Hsp90 (25, 40), their interactions with Hsp90 are
nonidentical.
Implications for Distinct Immunophilin Functions
Despite the overall similarity of FKBP51 and FKBP52, we have
observed in previous studies (10, 25, 26, 27, 29) and in the present study
that the two immunophilins interact with Hsp90 and with Hsp90-targeted
steroid receptors in distinctive manners. Collectively, these
observations make a compelling case for FKBP51 and FKBP52 having
functional distinctions. If so, then steroid receptor complexes
containing FKBP51 as opposed to FKBP52 may function differently.
Suggestive biological evidence for this comes from the altered ratio of
FKBPs observed in glucocorticoid-resistant squirrel monkey lymphoblasts
(24), and studies are currently underway to directly test the causal
relationship between elevated FKBP51/depressed FKBP52 levels in these
cells and reduced hormone binding affinity by GR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, rat Hsc70, human Hop, Hip, FKBP51, FKBP52,
and Cyp40) were prepared and used in a combined
transcription/translation system (TnT Lysate, Promega, Madison, WI) to
generate radiolabeled protein products as described in previous
publications (25, 28, 29, 30). The radiolabeled products were combined in
two ways with normal RL (1:1 lysate, Green Hectares, Oregon, WI)
supplemented with an ATP-regenerating system. One mixture contained
radiolabeled Hsp90, Hsp70, Hop, and Hip, and the other contained
radiolabeled Hsp90, FKBP51, FKBP52, and Cyp40. These mixtures (each 2
ml total) were used for the cell-free assembly of PR complexes as
described previously (2) with some modifications. Briefly, recombinant
chicken PR-A (
10 µg) was immunoaffinity purified from Sf9 cell
extracts using monoclonal antibody PR22 adsorbed to protein G-Sepharose
(Pharmacia, Piscataway, NJ). The PR-resin pellet was divided equally,
and RL assembly mixtures (prewarmed) were added to the pellets and
incubated at 30 C. Aliquots (200 µl, equivalent to approximately 0.5
µg PR) were removed from the assembly mixes at 1, 2, 3, 4, 5, 7, 10,
15, 30, and 60 min after initiation of assembly. Each aliquot was
immediately quenched in 1 ml ice-cold wash buffer (WB; 20
mM Tris, pH 7.4, 50 mM NaCl, 0.5% Tween 20) to
inhibit further assembly reactions. Resin pellets were washed four
times in cold WB and extracted with SDS sample buffer. Proteins were
separated by SDS-PAGE and visualized by Coomassie blue-staining and
autoradiography of the dried gel. Bands on the stained gel and x-ray
film were quantitated by laser scanning densitometry (Molecular
Dynamics, Sunnyvale, CA), and the absorbance values for radiolabeled
bands were normalized to the absorbance of stained PR-A in each
sample.
Two-Dimensional Gel Analysis of Steroid Receptor Complexes
Recombinant chicken PR-A and human ER- were immunoaffinity
purified from Sf9 insect cell extracts using monoclonal antibodies PR22
and Mab-17, respectively. GR was immunoaffinity purified from L-cell
extracts using monoclonal antibody BuGR-2 (Affinity Bioreagents,
Golden, CO). To assemble receptor complexes, approximately 0.5 µg of
each receptor (adsorbed to a 10-µl pellet of protein G-Sepharose and
10 µg antibody) was incubated with 500 µl RL plus an
ATP-regenerating system at 30 C for 45 min. Resin pellets were washed
four times in WB and extracted in isoelectric focusing (IEF) sample
solution. Proteins were separated in IEF dimension with ampholines in
the pH 3.5–10 range (Pharmacia). The second dimension SDS-PAGE gels
were silver stained to visualize protein spots.
General Approach to Production and in Vitro
Expression of Mutant cDNAs
Plasmids containing cDNAs encoding human FKBP51 and FKBP52 (25)
were used to produce more than 30 mutant and chimeric FKBP cDNAs for
these studies. Details on the generation of a few mutant plasmids are
given below. For other mutants, the sequences of oligonucleotide
mutagenic primers, conditions used for each mutagenesis reaction, and
procedures for subcloning of cDNA fragments will be provided on
request. In general, site-directed mutagenesis (QuickChange kit,
Stratagene, La Jolla, CA) was used to introduce: 1) stop codons for
C-terminal truncation mutants, 2) altered codons for point mutants, and
3) suitable restriction enzyme sites to facilitate creation of chimeric
FKBP cDNAs. Sequence changes in all mutated plasmids were confirmed by
automated sequencing.
Each of the plasmids was expressed in vitro by combined transcription/translation (TnT lysate, Promega) in the presence of [35S]methionine (DuPont/NEN, Boston, MA; specific activity, 1200 Ci/mmol). The synthesis of radiolabeled protein products was monitored by separation of 2 µl of each synthesis mixture by SDS-PAGE followed by autoradiography of the dried gel. Bands on x-ray film were quantitated by laser scanning densitometry (Molecular Dynamics) to determine relative levels of incorporated radioactivity.
Preparation of PPIase-Deficient FKBP51 Mutant
FKBP51/pET30, a bacterial expression plasmid that codes for
human FKBP51 with an N-terminal polyhistidine fusion (25), was mutated
at two adjacent, highly conserved codons in the PPIase domain. Using
site-directed mutagenesis, codon Phe-67 was changed to Asp, and Asp-68
was changed to Val; the alterations were verified by automated
sequencing of the mutant cDNA. Mutant and wild-type plasmids were
expressed in bacteria, recombinant proteins (FD67DV and wtFKBP51) were
purified from cell extracts, and proteins were assayed for PPIase
activity, as previously described (25).
Preparation of FKBP51/FKPBP52 Chimeras
The in vitro expression plasmid pSPUTK (Stratagene),
containing a cDNA insert for either human FKBP51 or human FKBP52, was
used for the preparation of chimeric cDNAs. Plasmids encoding chimeric
proteins (51C2 and 52C1) in which the C-terminal 66 amino acids of each
protein is swapped were generated by first using site-directed
mutagenesis to introduce a PvuII site overlapping codon 391
of FKBP51 cDNA. Taking advantage of the unique PvuII site at
the corresponding position (codon 393) in FKBP52’s cDNA and a pSPUTK
NheI site upstream of the insertion site for FKBP cDNAs,
plasmids were double-digested with NheI and
PvuII. After separation of digestion products on agarose
gels, the NheI/PvuII fragments were religated to
the alternate plasmid-containing fragments.
Plasmids encoding 51T2 and 52T1, chimeric proteins in which the TPR domains have been swapped, were generated as follows. An AccI restriction enzyme site was introduced by site-directed mutagenesis at codon 279 of wtFKBP52; this site corresponds to an endogenous AccI site in wtFKBP51. Using the FKBP51 plasmid with a PvuII site introduced at codon 391, both plasmids were double-digested with AccI and PvuII; the resulting fragments were isolated from agarose gels and cross-ligated to produce cDNAs encoding the chimeric proteins.
A plasmid encoding 52V1, an FKBP52 with the C-terminal 53 amino acids of FKBP51, was produced by taking advantage of unique SmaI(Arg-406) and SapI (Glu-443) restriction sites in wtFKBP52 cDNA and a unique SapI site in the 3'-untranslated region of FKBP51 cDNA. A PCR product was generated using a high-fidelity polymerase (Deep Vent Exo+, New England Biolabs, Beverly, MA) wtFKBP51 cDNA as template, a forward primer (5'-TATACCCGGGACCGCAGGAT-ATACGC) introducing a SmaI site at codon 404, and a reverse primer (5'-ATATGCTCTTCTG-CTTCCAGAATCACATAGC) overlapping the SapI site. The FKBP51 PCR product and the FKBP52 plasmid were both digested with SmaI and SapI, and the appropriate gel bands were religated.
Plasmids encoding chimeric proteins 51Tbc2 and 52Tbc1, in which the second and third TPR units are swapped between FKBPs, were generated from the earlier chimeric constructs 51C2 and 52C1. The latter plasmids were digested with NheI, a pSPUTK site upstream from the insertion site, and MscI, a site within the coding region for the second TPR unit in both FKBPs. The digestion products were separated by gel electrophoresis, and the NheI/MscI fragments were religated to the alternate plasmid-containing fragments.
Interactions of FKBP Forms with PR and Hsp90
PR and Hsp90 complexes were assembled in vitro using
RL supplemented with radiolabeled FKBP forms. In some cases, equimolar
amounts of FKBP forms were included; in others, FKBP52 forms were
included at a 5-fold molar excess over FKBP51 forms to reflect the
ratio of endogenous FKBPs in RL. Relative molar quantities were
estimated after quantification of incorporated radioactivity in each
synthesis mixture with adjustments for the methionine content of each
product.
Recombinant cPR-A immobilized on immunoaffinity resin was added to RL mixtures and incubated at 30 C for 30 min. Each sample contained 0.5 µg PR protein on a 10-µl resin pellet and 300 µl RL mixture containing an ATP-regenerating system and one of the radiolabeled FKBP forms. Resin pellets were washed four times in cold WB and extracted with SDS sample buffer. Proteins were separated by SDS-PAGE and visualized by Coomassie staining and autoradiography of the dried gel.
Hsp90 complexes were similarly analyzed. The anti-Hsp90 monoclonal antibody H90–10 was preadsorbed to protein A-Sepharose (Pharmacia), and resin was aliquoted into separate tubes (10 µg antibody on a 10-µl resin pellet per tube). Each sample was incubated with 100 µl RL mixture before gel separations and analysis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grant R01–48218.
Received for publication October 24, 1997. Revision received December 9, 1997. Accepted for publication December 11, 1997.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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  Y. Hirota, S. Tranguch, T. Daikoku, A. Hasegawa, Y. Osuga, Y. Taketani, and S. K. Dey Deficiency of Immunophilin FKBP52 Promotes Endometriosis Am. J. Pathol., December 1, 2008; 173(6): 1747 - 1757. [Abstract] [Full Text] [PDF]
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 D. F. Smith and D. O. Toft Minireview: The Intersection of Steroid Receptors with Molecular Chaperones: Observations and Questions Mol. Endocrinol., October 1, 2008; 22(10): 2229 - 2240. [Abstract] [Full Text] [PDF]
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 A. K. McCollum, K. B. Lukasiewicz, C. J. TenEyck, W. L. Lingle, D. O. Toft, and C. Erlichman Cisplatin abrogates the geldanamycin-induced heat shock response Mol. Cancer Ther., October 1, 2008; 7(10): 3256 - 3264. [Abstract] [Full Text] [PDF]
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 A. K. McCollum, C. J. TenEyck, B. Stensgard, B. W. Morlan, K. V. Ballman, R. B. Jenkins, D. O. Toft, and C. Erlichman P-Glycoprotein-Mediated Resistance to Hsp90-Directed Therapy Is Eclipsed by the Heat Shock Response Cancer Res., September 15, 2008; 68(18): 7419 - 7427. [Abstract] [Full Text] [PDF]
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 D. L. Riggs, M. B. Cox, H. L. Tardif, M. Hessling, J. Buchner, and D. F. Smith Noncatalytic Role of the FKBP52 Peptidyl-Prolyl Isomerase Domain in the Regulation of Steroid Hormone Signaling Mol. Cell. Biol., December 15, 2007; 27(24): 8658 - 8669. [Abstract] [Full Text] [PDF]
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M. B. Cox, D. L. Riggs, M. Hessling, F. Schumacher, J. Buchner, and D. F. Smith FK506-Binding Protein 52 Phosphorylation: A Potential Mechanism for Regulating Steroid Hormone Receptor Activity Mol. Endocrinol., December 1, 2007; 21(12): 2956 - 2967. [Abstract] [Full Text] [PDF]
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 B. Chambraud, H. Belabes, V. Fontaine-Lenoir, A. Fellous, and E. E. Baulieu The immunophilin FKBP52 specifically binds to tubulin and prevents microtubule formation FASEB J, September 1, 2007; 21(11): 2787 - 2797. [Abstract] [Full Text] [PDF]
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K. Y. Lee, J.-W. Jeong, J. Wang, L. Ma, J. F. Martin, S. Y. Tsai, J. P. Lydon, and F. J. DeMayo Bmp2 Is Critical for the Murine Uterine Decidual Response Mol. Cell. Biol., August 1, 2007; 27(15): 5468 - 5478. [Abstract] [Full Text] [PDF]
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 A. J. Bredemeyer, P. E. Carrigan, T. A. Fehniger, D. F. Smith, and T. J. Ley Hop Cleavage and Function in Granzyme B-induced Apoptosis J. Biol. Chem., December 1, 2006; 281(48): 37130 - 37141. [Abstract] [Full Text] [PDF]
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A. K. McCollum, C. J. TenEyck, B. M. Sauer, D. O. Toft, and C. Erlichman Up-regulation of Heat Shock Protein 27 Induces Resistance to 17-Allylamino-Demethoxygeldanamycin through a Glutathione-Mediated Mechanism. Cancer Res., November 15, 2006; 66(22): 10967 - 10975. [Abstract] [Full Text] [PDF]
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B. R. Keppler, A. T. Grady, and M. B. Jarstfer The Biochemical Role of the Heat Shock Protein 90 Chaperone Complex in Establishing Human Telomerase Activity J. Biol. Chem., July 21, 2006; 281(29): 19840 - 19848. [Abstract] [Full Text] [PDF]
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 K.-Y. Kim, K.-C. Choi, N. Auersperg, and P. C K Leung Mechanism of gonadotropin-releasing hormone (GnRH)-I and -II-induced cell growth inhibition in ovarian cancer cells: role of the GnRH-I receptor and protein kinase C pathway. Endocr. Relat. Cancer, March 1, 2006; 13(1): 211 - 220. [Abstract] [Full Text] [PDF]
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S. J. H. Arlander, S. J. Felts, J. M. Wagner, B. Stensgard, D. O. Toft, and L. M. Karnitz Chaperoning Checkpoint Kinase 1 (Chk1), an Hsp90 Client, with Purified Chaperones J. Biol. Chem., February 3, 2006; 281(5): 2989 - 2998. [Abstract] [Full Text] [PDF]
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 J. A. Magee, L.-w. Chang, G. D. Stormo, and J. Milbrandt Direct, Androgen Receptor-Mediated Regulation of the FKBP5 Gene via a Distal Enhancer Element Endocrinology, January 1, 2006; 147(1): 590 - 598. [Abstract] [Full Text] [PDF]
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 S. Tranguch, J. Cheung-Flynn, T. Daikoku, V. Prapapanich, M. B. Cox, H. Xie, H. Wang, S. K. Das, D. F. Smith, and S. K. Dey From The Cover: Cochaperone immunophilin FKBP52 is critical to uterine receptivity for embryo implantation PNAS, October 4, 2005; 102(40): 14326 - 14331. [Abstract] [Full Text] [PDF]
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W. B. Denny, V. Prapapanich, D. F. Smith, and J. G. Scammell Structure-Function Analysis of Squirrel Monkey FK506-Binding Protein 51, a Potent Inhibitor of Glucocorticoid Receptor Activity Endocrinology, July 1, 2005; 146(7): 3194 - 3201. [Abstract] [Full Text] [PDF]
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L. Wang, T. V. Nguyen, R. W. McLaughlin, L. A. Sikkink, M. Ramirez-Alvarado, and R. M. Weinshilboum Human thiopurine S-methyltransferase pharmacogenetics: Variant allozyme misfolding and aggresome formation PNAS, June 28, 2005; 102(26): 9394 - 9399. [Abstract] [Full Text] [PDF]
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 R. K. Ramanathan, D. L. Trump, J. L. Eiseman, C. P. Belani, S. S. Agarwala, E. G. Zuhowski, J. Lan, D. M. Potter, S. P. Ivy, S. Ramalingam, et al. Phase I Pharmacokinetic-Pharmacodynamic Study of 17-(Allylamino)-17-Demethoxygeldanamycin (17AAG, NSC 330507), a Novel Inhibitor of Heat Shock Protein 90, in Patients with Refractory Advanced Cancers Clin. Cancer Res., May 1, 2005; 11(9): 3385 - 3391. [Abstract] [Full Text] [PDF]
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T. Daikoku, S. Tranguch, D. B. Friedman, S. K. Das, D. F. Smith, and S. K. Dey Proteomic Analysis Identifies Immunophilin FK506 Binding Protein 4 (FKBP52) as a Downstream Target of Hoxa10 in the Periimplantation Mouse Uterus Mol. Endocrinol., March 1, 2005; 19(3): 683 - 697. [Abstract] [Full Text] [PDF]
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 M. P. Goetz, D. Toft, J. Reid, M. Ames, B. Stensgard, S. Safgren, A. A. Adjei, J. Sloan, P. Atherton, V. Vasile, et al. Phase I Trial of 17-Allylamino-17-Demethoxygeldanamycin in Patients With Advanced Cancer J. Clin. Oncol., February 20, 2005; 23(6): 1078 - 1087. [Abstract] [Full Text] [PDF]
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G. M. Wochnik, J. Ruegg, G. A. Abel, U. Schmidt, F. Holsboer, and T. Rein FK506-binding Proteins 51 and 52 Differentially Regulate Dynein Interaction and Nuclear Translocation of the Glucocorticoid Receptor in Mammalian Cells J. Biol. Chem., February 11, 2005; 280(6): 4609 - 4616. [Abstract] [Full Text] [PDF]
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B. Wu, P. Li, Y. Liu, Z. Lou, Y. Ding, C. Shu, S. Ye, M. Bartlam, B. Shen, and Z. Rao 3D structure of human FK506-binding protein 52: Implications for the assembly of the glucocorticoid receptor/Hsp90/immunophilin heterocomplex PNAS, June 1, 2004; 101(22): 8348 - 8353. [Abstract] [Full Text] [PDF]
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J. Botos, W. Xian, D. F. Smith, and C. L. Smith Progesterone Receptor Deficient in Chromatin Binding Has an Altered Cellular State J. Biol. Chem., April 9, 2004; 279(15): 15231 - 15239. [Abstract] [Full Text] [PDF]
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 Z. He, L. Li, and S. Luan Immunophilins and Parvulins. Superfamily of Peptidyl Prolyl Isomerases in Arabidopsis Plant Physiology, April 1, 2004; 134(4): 1248 - 1267. [Abstract] [Full Text] [PDF]
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S. J. H. Arlander, A. K. Eapen, B. T. Vroman, R. J. McDonald, D. O. Toft, and L. M. Karnitz Hsp90 Inhibition Depletes Chk1 and Sensitizes Tumor Cells to Replication Stress J. Biol. Chem., December 26, 2003; 278(52): 52572 - 52577. [Abstract] [Full Text] [PDF]
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M. Tesic, J. A. Marsh, S. B. Cullinan, and R. F. Gaber Functional Interactions between Hsp90 and the Co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae J. Biol. Chem., August 29, 2003; 278(35): 32692 - 32701. [Abstract] [Full Text] [PDF]
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T. R. Hubler, W. B. Denny, D. L. Valentine, J. Cheung-Flynn, D. F. Smith, and J. G. Scammell The FK506-Binding Immunophilin FKBP51 Is Transcriptionally Regulated by Progestin and Attenuates Progestin Responsiveness Endocrinology, June 1, 2003; 144(6): 2380 - 2387. [Abstract] [Full Text] [PDF]
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J. Cheung-Flynn, P. J. Roberts, D. L. Riggs, and D. F. Smith C-terminal Sequences outside the Tetratricopeptide Repeat Domain of FKBP51 and FKBP52 Cause Differential Binding to Hsp90 J. Biol. Chem., May 2, 2003; 278(19): 17388 - 17394. [Abstract] [Full Text] [PDF]
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C. R. Sinars, J. Cheung-Flynn, R. A. Rimerman, J. G. Scammell, D. F. Smith, and J. Clardy Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes PNAS, February 4, 2003; 100(3): 868 - 873. [Abstract] [Full Text] [PDF]
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 W. B. Pratt and D. O. Toft Regulation of Signaling Protein Function and Trafficking by the hsp90/hsp70-Based Chaperone Machinery Experimental Biology and Medicine, February 1, 2003; 228(2): 111 - 133. [Abstract] [Full Text] [PDF]
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B. K. Ward, R. K. Allan, D. Mok, S. E. Temple, P. Taylor, J. Dornan, P. J. Mark, D. J. Shaw, P. Kumar, M. D. Walkinshaw, et al. A Structure-based Mutational Analysis of Cyclophilin 40 Identifies Key Residues in the Core Tetratricopeptide Repeat Domain That Mediate Binding to Hsp90 J. Biol. Chem., October 18, 2002; 277(43): 40799 - 40809. [Abstract] [Full Text] [PDF]
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A. Kazlauskas, L. Poellinger, and I. Pongratz Two Distinct Regions of the Immunophilin-like Protein XAP2 Regulate Dioxin Receptor Function and Interaction with hsp90 J. Biol. Chem., March 29, 2002; 277(14): 11795 - 11801. [Abstract] [Full Text] [PDF]
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 W. Zhu, J.-S. Zhang, and C. Y.F. Young Silymarin inhibits function of the androgen receptor by reducing nuclear localization of the receptor in the human prostate cancer cell line LNCaP Carcinogenesis, September 1, 2001; 22(9): 1399 - 1403. [Abstract] [Full Text] [PDF]
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W. B. Denny, D. L. Valentine, P. D. Reynolds, D. F. Smith, and J. G. Scammell Squirrel Monkey Immunophilin FKBP51 Is a Potent Inhibitor of Glucocorticoid Receptor Binding Endocrinology, November 1, 2000; 141(11): 4107 - 4113. [Abstract] [Full Text] [PDF]
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J. Cheung and D. F. Smith Molecular Chaperone Interactions with Steroid Receptors: an Update Mol. Endocrinol., July 1, 2000; 14(7): 939 - 946. [Full Text]
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S. J. Felts, B. A. L. Owen, P. Nguyen, J. Trepel, D. B. Donner, and D. O. Toft The hsp90-related Protein TRAP1 Is a Mitochondrial Protein with Distinct Functional Properties J. Biol. Chem., February 4, 2000; 275(5): 3305 - 3312. [Abstract] [Full Text] [PDF]
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A. M. Silverstein, M. D. Galigniana, K. C. Kanelakis, C. Radanyi, J.-M. Renoir, and W. B. Pratt Different Regions of the Immunophilin FKBP52 Determine Its Association with the Glucocorticoid Receptor, hsp90, and Cytoplasmic Dynein J. Biol. Chem., December 24, 1999; 274(52): 36980 - 36986. [Abstract] [Full Text] [PDF]
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 S. E. Holt, D. L. Aisner, J. Baur, V. M. Tesmer, M. Dy, M. Ouellette, J. B. Trager, G. B. Morin, D. O. Toft, J. W. Shay, et al. Functional requirement of p23 and Hsp90 in telomerase complexes Genes & Dev., April 1, 1999; 13(7): 817 - 826. [Abstract] [Full Text]
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D. F. Nathan, M. H. Vos, and S. Lindquist Identification of SSF1, CNS1, and HCH1 as multicopy suppressors of a Saccharomyces cerevisiae Hsp90 loss-of-function mutation PNAS, February 16, 1999; 96(4): 1409 - 1414. [Abstract] [Full Text] [PDF]
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 P. D. Reynolds, Y. Ruan, D. F. Smith, and J. G. Scammell Glucocorticoid Resistance in the Squirrel Monkey Is Associated with Overexpression of the Immunophilin FKBP51 J. Clin. Endocrinol. Metab., February 1, 1999; 84(2): 663 - 669. [Abstract] [Full Text]
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A. Carrello, E. Ingley, R. F. Minchin, S. Tsai, and T. Ratajczak The Common Tetratricopeptide Repeat Acceptor Site for Steroid Receptor-associated Immunophilins and Hop Is Located in the Dimerization Domain of Hsp90 J. Biol. Chem., January 29, 1999; 274(5): 2682 - 2689. [Abstract] [Full Text] [PDF]
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S. Chen and D. F. Smith Hop as an Adaptor in the Heat Shock Protein 70 (Hsp70) and Hsp90 Chaperone Machinery J. Biol. Chem., December 25, 1998; 273(52): 35194 - 35200. [Abstract] [Full Text] [PDF]
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J. A. Marsh, H. M. Kalton, and R. F. Gaber Cns1 Is an Essential Protein Associated with the Hsp90 Chaperone Complex in Saccharomyces cerevisiae That Can Restore Cyclophilin 40-Dependent Functions in cpr7Delta Cells Mol. Cell. Biol., December 1, 1998; 18(12): 7353 - 7359. [Abstract] [Full Text]
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 D. F. Smith, L. Whitesell, and E. Katsanis Molecular Chaperones: Biology and Prospects for Pharmacological Intervention Pharmacol. Rev., December 1, 1998; 50(4): 493 - 514. [Abstract] [Full Text] [PDF]
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H. L. Forsythe, J. L. Jarvis, J. W. Turner, L. W. Elmore, and S. E. Holt Stable Association of hsp90 and p23, but Not hsp70, with Active Human Telomerase J. Biol. Chem., May 4, 2001; 276(19): 15571 - 15574. [Abstract] [Full Text] [PDF]
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D. R. Bell and A. Poland Binding of Aryl Hydrocarbon Receptor (AhR) to AhR-interacting Protein. THE ROLE OF hsp90 J. Biol. Chem., November 10, 2000; 275(46): 36407 - 36414. [Abstract] [Full Text] [PDF]
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M. D. Galigniana, C. Radanyi, J.-M. Renoir, P. R. Housley, and W. B. Pratt Evidence That the Peptidylprolyl Isomerase Domain of the hsp90-binding Immunophilin FKBP52 Is Involved in Both Dynein Interaction and Glucocorticoid Receptor Movement to the Nucleus J. Biol. Chem., April 27, 2001; 276(18): 14884 - 14889. [Abstract] [Full Text] [PDF]
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F. Pirkl, E. Fischer, S. Modrow, and J. Buchner Localization of the Chaperone Domain of FKBP52 J. Biol. Chem., September 28, 2001; 276(40): 37034 - 37041. [Abstract] [Full Text] [PDF]
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