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Mol Cell Biol, February 1998, p. 944-952, Vol. 18, No. 2
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mutation of Hip's Carboxy-Terminal Region Inhibits
a Transitional Stage of Progesterone Receptor Assembly
Viravan
Prapapanich,
Shiying
Chen, and
David F.
Smith*
Department of Pharmacology, University of
Nebraska Medical Center, Omaha, Nebraska 68198-6260
Received 9 September 1997/Returned for modification 21 October
1997/Accepted 14 November 1997
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ABSTRACT |
Steroid receptor complexes are assembled through an ordered,
multistep pathway involving multiple components of the cytoplasmic chaperone machinery. Two of these components are Hsp70-binding proteins, Hip and Hop, that have some limited homology in their C-terminal regions, outside the sequences mapped for Hsp70 binding. Within this region of Hip is a DPEV sequence that occurs twice; in Hop,
one DPEV sequence plus a partial second sequence occurs. In an effort
to better understand Hip function as it relates to assembly of
progesterone receptor complexes, the DPEV region of Hip was targeted
for mutations. Each DPEV sequence was mutated to an APAV sequence,
singly or in combination. The combined mutation, APAV2, was
further combined with a deletion of Hip's tetratricopeptide repeat
region that is required for Hsp70 binding or with a deletion of Hip's
GGMP repeat. An additional mutant was prepared by truncation of Hip's
DPEV-containing C terminus. By comparing interactions of various Hip
forms with Hsp70, it was determined that mutation of the DPEV sequences
created a dominant inhibitory form of Hip. The mutant Hip-Hsp70 complex
was not prevented from interacting with progesterone receptor, but the
mutant caused a dose-dependent inhibition of receptor assembly with
Hsp90. The behavior of the Hip mutant is consistent with a model in
which Hip and Hop are required to facilitate the transition from an
early receptor complex with Hsp70 into later complexes containing
Hsp90.
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INTRODUCTION |
Prior to binding hormone, steroid
receptor monomers are typically found in a multiprotein complex
containing Hsp90 and Hsp90-associated proteins such as p23 and large
immunophilins (for an extensive review, see reference
21). Receptors for progesterone (PR) and glucocorticoids (GR) must be assembled in these complexes in order to
bind hormone with high affinity and efficiency. Studies of the receptor
assembly process, relying primarily on cell-free reactions in rabbit
reticulocyte lysate (RL), have revealed that assembly is an ordered
process and involves at least eight proteins that are components of the
molecular chaperone machinery. Several of these chaperone components
appear transiently prior to formation of mature receptor complexes. For
example, Hsp70 is the first protein observed to bind PR in a cell-free
assembly, but Hsp70 is probably not a component of mature complexes
(1, 25). Two Hsp70-associated proteins, Hip and Hop, are
recovered in PR complexes shortly after the first appearance of Hsp70,
but these proteins are also absent from mature complexes. A model in
which Hip and Hop function in a coordinated manner to facilitate
Hsp70-mediated folding of proteins has been proposed (8),
but it is not clear how this relates to steroid receptor assembly.
Hop (alternate names in the literature are p60, IEF SSP 3521, mSti1,
and RF-hsp70) is required for formation of mature PR (4) and
GR (5, 6) complexes. In a yeast model system, deletion of
the gene for Sti1, a Saccharomyces cerevisiae homolog for
Hop, resulted in decreased function of heterologously expressed vertebrate GR (3). Hop binds Hsp70 through an N-terminal
tetratricopeptide repeat (TPR) region, but it can simultaneously bind
Hsp90 through an internal TPR (4, 17). Hop appears to be
quantitatively associated with Hsp90, but there is typically a
substoichiometric amount of Hsp70 in immunoaffinity-purified Hop
complexes (24). In one report, evidence that Hop catalyzes
the dissociation of ADP from Hsp70 in exchange for ATP was presented
(11), but the absence of contaminating DnaJ or other
activities in the protein preparations was not demonstrated.
Less is known about the functional requirement for Hip in the receptor
assembly pathway. Hip was first noted as a transient component during
the cell-free assembly of PR complexes (25) and was
subsequently found to be associated with Hsp70 (19). In a
screen for proteins interacting with the ATPase domain of Hsp70, Hip
was identified and shown to stabilize binding of Hsp70 to a misfolded
protein substrate (12). Hip binds Hsp70 in an ADP-dependent
manner (12) through a central TPR motif (15, 20).
There is no apparent homolog for Hip in the S. cerevisiae genomic database. Since vertebrate steroid receptors function in yeast,
this would argue against an absolute need for Hip, but it is possible
that Hip's function is fulfilled by some other factor in yeast.
In designing Hip mutants, we chose to target the C-terminal region of
Hip that shows some limited homology with the C-terminal region of Hop.
In this report, mutations in this region of Hip are shown to generate
dominant inhibitory forms of Hip that block in vitro assembly of Hop
and Hsp90 with PR complexes.
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MATERIALS AND METHODS |
In vitro expression plasmids.
A QuikChange site-directed
mutagenesis kit (Stratagene) was used to substitute alanines for the
aspartic and glutamic acid residues in the DPEV sequences, beginning at
codons 318 and 327, contained in Hip's C-terminal region. A 1.4-kb
cDNA containing the open reading frame and 3' untranslated region of
the human Hip gene (19) was subcloned into pSPUTK plasmid
(Stratagene) for construction of APAV mutants. Sequences for the
mutagenic oligonucleotides targeting the first and second DPEV
sequences, respectively, were
5'-GAAATTCTTAGTGCTCCAGCGGTTCTTGCAGCCATG
and 5'-CATGGCTGCAAGAACCGCTGGAGCACTAAGAATTTC
(the mutated bases are underlined). Mutants were created for each
DPEV alone (APAV-a and APAV-b) and for the two DPEV sites in
combination (APAV2).
The generation of plasmids encoding 
GGMP, TPR, and N-303 was
described previously (20). To create combinations of
APAV2 with TPR (TAP) and GGMP (GAP), the
mutated region of the APAV2 cDNA was substituted into the
corresponding position of the deletion mutants by using a unique
SphI restriction enzyme site.
The preparation of a plasmid encoding the ATPase domain of rat Hsc70
(Hsc70-AD) was described previously (
20). A plasmid
encoding
rat Hsc70's peptide-binding domain (Hsc70-PD) was prepared
by first
using site-directed mutagenesis to introduce an
NcoI
site at
codon 379. The resulting cDNA, containing an
EcoRI site
at
the 3' cloning site, was digested with
NcoI and
EcoRI and subcloned
into pSPUTK. The point mutant K71E was
prepared by site-directed
mutagenesis of codon 71 of rat Hsc70 cDNA
cloned into pSPUTK.
The sequences of all mutated plasmids were
confirmed by automated
sequencing.
All plasmids were expressed in vitro in a combined
transcription-translation system (TnT Lysate; Promega) according to the
guidelines suggested by the manufacturer. Protein products were
radiolabeled by inclusion of [
35S]methionine (DuPont/NEN;
specific activity, 1,200 Ci/mmol) in
the synthesis mixture. Two
microliters of each synthesis mixture
was separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and the dried
gel was autoradiographed. Bands were
quantitated by laser scanning
densitometry (Molecular Dynamics).
After adjustment for the number of
methionines in each product,
equivalent molar amounts of radiolabeled
products could be determined.
Bacterial expression and purification of recombinant Hip
forms.
Expression and purification of Hip and mutants involved
generating a fusion protein (Hip-intein/CBD) in which Hip's C terminus is contiguous with a self-cutting intein and chitin-binding domain (CBD). First, a PCR product was generated by using Hip/pSPUTK as a
template, a forward oligonucleotide primer complementary to the 5'
pSPUTK cloning site (5'-GCAGAAGCTCAGAATAAACGC), and a
reverse primer complementary to the final seven codons of the open
reading frame for Hip (5'-CGCTTGACCTCCAAATTTGGC). A
high-fidelity polymerase (Deep Vent Exo+; New England
Biolabs) was used for 25 cycles of amplification. The PCR product was
digested with NcoI and subcloned into pCYB4 (Impact I kit;
New England Biolabs) that had been digested with NcoI and
SmaI. Automated sequencing was performed to verify the correct sequence of the entire cloned PCR product.
To enhance expression of the Hip-intein/CBD fusion protein in bacteria,
the region coding for the fusion protein was removed
from pCYB4 and
subcloned into pET28 (Novagen). With the Hip-intein/CBD/pET28
plasmid
as the host, mutant Hip-intein/CBD constructs were prepared
by
replacing the wild-type Hip sequence with mutant Hip cDNA sequences.
Transformation of BL21 bacterial cells, expression of protein,
and cell
extract preparation were performed as described previously
(
20).
Proteins were purified from bacterial extracts by using reagents and
instructions supplied with the Impact I kit. Briefly,
fusion proteins
were bound to a chitin affinity resin. After being
washed, the resin
was incubated overnight with reducing agent
to promote intein-mediated
cleavage of Hip proteins from the fusion
product. The cleaved Hip
products were collected in 20 mM Tris-HCl-50
mM NaCl-0.1 mM EDTA and
concentrated by using Centriprep 30 devices
(Amicon). The C terminus of
each product differed from that of
native Hip by the addition of a
single glycine residue.
Interaction of Hip forms with Hsp70.
The abilities of Hip
forms to bind Hsp70 immobilized on an immunoaffinity resin were
assessed essentially as described previously (20). Briefly,
anti-Hsp70 monoclonal antibody BB70 was adsorbed to protein G-Sepharose
(Pharmacia) and used to immunoprecipitate Hsp70 from rabbit RL (1:1;
from Green Hectares, Oregon, Wis.) that was supplemented with a
radiolabeled Hip form. Each sample contained the same molar equivalent
of radiolabeled Hip or Hip mutant in 100 µl of RL with 15 µg of
BB70 on a 10-µl resin pellet. All samples were incubated at 30°C
for 30 min. The resin pellets were divided and washed three times with
wash buffer (WB) (20 mM Tris [pH 7.4], 50 mM NaCl, 10 mM
monothioglycerol, 0.5% Tween 20) or WB plus 0.5 M NaCl. Proteins
adsorbed to resin were separated by SDS-PAGE and visualized by
Coomassie blue staining and autoradiography of the dried gel.
In an alternate set of experiments to compare interactions of wild-type
Hip (wtHip) and APAV
2 with Hsp70, purified recombinant Hip
forms were first adsorbed
to an immunoaffinity resin. The resin was
prepared by binding
the anti-Hip monoclonal antibody 2G6
(
19) to protein A-Sepharose
(Pharmacia). To avoid later
contamination by antibody heavy chain
in the gel migration region for
Hip, 2G6 was covalently coupled
to resin according to a published
procedure (
23). Radiolabeled
rat Hsc70 or mutant K71E was
prepared by transcription-translation
(TnT kit; Promega) of in vitro
expression plasmids in the presence
of [
35S]methionine. A
10-µl pellet of immunoaffinity resin containing
10 µg of wtHip or
APAV
2 was added to 100 µl of rabbit RL (Green Hectares)
supplemented
with radiolabeled Hsc70 or K71E. Resin complexes were
incubated
at 30°C for 30 min, washed with WB, and separated by
SDS-PAGE
for Coomassie blue staining and autoradiography.
Radiolabeled Hsc70-AD and Hsp70-PD were prepared by in vitro
transcription-translation of the appropriate plasmids. To distinguish
interactions of Hip forms with the separate Hsp70 domains, an
aliquot
of each radiolabeled synthesis mixture was added to 100
µl of
incubation buffer (20 mM Tris-HCl, 10 mM monothioglycerol,
50 mM NaCl,
5 mM ADP, 5 mM MgCl
2, and 0.5% Tween 20) containing
wtHip
or APAV
2 resin complexes. Samples were incubated at 30°C
for 30 min, divided
equally, and washed with either WB alone or WB plus
0.5 M NaCl.
Protein components on resins were separated by SDS-PAGE and
visualized
by Coomassie blue staining and autoradiography.
PR reconstitution reactions.
With some minor modifications,
PR assembly reactions were performed as described previously
(27). Briefly, individual reactions were performed in 100 to
200 µl of RL supplemented with an ATP-regenerating system and
containing approximately 1 µg of recombinant chicken PR-A bound to 15 µg of monoclonal antibody PR22 on a 10-µl pellet of protein G- or
protein A-Sepharose. The resins were incubated for 30 to 45 min at
30°C, washed four times with WB, and extracted with SDS sample
buffer.
In one set of reactions, RL was immunodepleted of endogenous Hip in the
following manner. In the first depletion step, 100
µg of monoclonal
antibody 3A4, which recognizes only free Hip
(
19), was
adsorbed to a 15-µl pellet of protein G-Sepharose
and gently rocked
with 1 ml of RL at 4°C for 8 h. The supernatant
from this step
was retreated with a combination of anti-Hip antibodies
(3A4, 2G6, and
10D1; 100 µg total) for an additional 8 h at 4°C.
Mock-depleted RL was prepared by two treatments with PR22. The
efficiency of Hip depletion was estimated by quantifying
Western-immunostained
bands from 2 µl of mock-depleted or
Hip-depleted lysates. PR reconstitutions
were performed with either
mock-depleted or Hip-depleted lysates,
and the relative recovery of Hip
in PR complexes was quantitated
by densitometry of Western-stained
bands.
In another set of experiments, purified recombinant Hip forms were
added to RL for use in PR assembly reactions. Either wtHip
or mutant
Hip forms were added to RL at final concentrations as
high as 12 µM
(approximately a 12-fold molar excess over endogenous
Hip levels). In
control experiments, up to 32 µM
-casein or reduced
carboxymethylated lactalbumin (RCMLA) (Sigma) was added in place
of Hip
forms. PR assembly reactions were performed as usual.
Dynamics of Hip and Hsp70 interactions.
Pulse-chase-type
experiments were performed to monitor the exchange of Hsp70 in PR
complexes or in Hip-Hsp70 complexes. In the PR experiment, complexes
were assembled in two phases. In both phases, 800 µl of RL which
contained an ATP-regenerating system and 20 µg of geldanamycin (GA)
per ml to enhance formation of early and intermediate PR complexes
containing Hsp70 and Hip was used (27). Also in both phases,
the RL was supplemented with a 10-fold molar excess of purified wtHip,
APAV2, or N-303. In the first assembly phase only, samples
contained radiolabeled Hsp70. After steady-state assembly conditions
were established in the first phase (30 min at 30°C), PR-resin
complexes were transferred to fresh RL prewarmed to 30°C for the
second assembly phase. At 0, 1, 2, 3, 5, 10, 15, and 25 min, 100-µl
aliquots were removed from the second-assembly mixtures and immediately quenched with 1 ml of cold WB to inhibit further assembly reactions. As
a negative control, a parallel set of samples with PR22 resin lacking
PR-A was used. All samples were separated by SDS-PAGE, Coomassie blue
stained, and autoradiographed. Stained protein bands and bands on X-ray
film were quantitated by densitometry.
To measure exchange of Hsp70 on immobilized Hip forms, recombinant
intein-CBD fusion proteins (wtHip, APAV
2, or
TAP) were
preadsorbed to chitin resin. In a similar experiment,
His-tagged wtHip
or mutant N-303 was adsorbed to Ni
2+-resin. Hip forms on
resin (42 µg of protein on a 70-µl resin
pellet) or
Ni
2+-resin alone was separately added to 700 µl of RL
supplemented
with an ATP-regenerating system and 5 to 8 µl of
radiolabeled
Hsp70 synthesis mixture. After a 30-min incubation at
30°C, the
resins were transferred to fresh, prewarmed RL lacking
radiolabeled
Hsp70. As with PR complexes, aliquots were removed at
various
times and quenched in cold WB. Samples were separated by
SDS-PAGE,
Coomassie blue stained, and autoradiographed.
Hop binding to purified Hsp70.
Hop was immunoaffinity
purified from RL that was adjusted to 0.4 M NaCl to dissociate Hop
complexes containing Hsp70 and Hsp90. Each sample contained 10 µg of
monoclonal antibody F5 adsorbed to a 10-µl pellet of protein
A-Sepharose (Pharmacia) and approximately 1 µg of immunoadsorbed Hop.
Reaction mixtures (100 µl, final volume) were prepared by combining
70 nM purified bovine Hsc70 (StressGen) alone or with 700 nM
-casein, RCMLA, or bovine serum albumin (BSA) in buffer (20 mM Tris
[pH 7.4], 50 mM KCl, 5 mM ADP, 5 mM MgCl2, 10 mM
monothioglycerol, and 0.5% Tween 20) and preincubating the mixture at
30°C for 15 min. Each reaction mixture was then added to Hop or
control resin pellets and incubated for an additional 20 min at 30°C.
The resins were washed four times in cold WB, and bound proteins were
extracted into SDS sample buffer for gel separations.
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RESULTS |
It was noted previously that the C-terminal region of Hip shows
some limited homology with Hop (12), although this region does not appear to be required for known protein interactions of either
Hip (15, 20) or Hop (4, 17). In Fig.
1A, the sequences of Hip and Hop are
diagramed with the amino acid sequences of their C termini aligned. The
two DPEV sequences in the C terminus of Hip were selected as target
sites for mutagenesis.
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FIG. 1.
(A) Comparison of Hip and Hop sequences. Hsp70-binding
proteins Hip and Hop both contain TPR domains. In Hip, the TPR domain
plus surrounding highly charged sequences is required for Hsp70
binding. Also contained in Hip is a GGMP repeat domain (G) whose
function is unknown. Hop contains two TPR regions; the N-terminal one
is required for Hsp70 binding, and the central TPR binds to Hsp90.
Little homology is shared between the highly degenerate TPR regions of
Hip and Hop. The region of greatest similarity is near the C terminus
of each protein (amino acid sequence alignment in exploded view at
bottom). Most notable in this region are two DPEV sequences in Hip that
align with a DPEV and DPAM in Hop (highlighted by shaded boxes). (B)
Hip mutants. Each of the DPEV sequences in Hip was mutated to APAV, as
illustrated. The mutation of both DPEV sequences (APAV2)
was also combined with previously developed mutants in which the TPR or
GGMP domains had been deleted. Another mutant from previous studies is
the truncation mutant N-303, which lacks the DPEV-containing C terminus
of Hip. TPR domains (open boxes), highly charged regions (+ +), and
a region of Hop homology (shaded box) at the C terminus are shown.
a.a., amino acids; WT, wild type.
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Expression plasmids encoding each of the Hip mutants diagramed in Fig.
1B were generated. The two DPEV sequences were mutated to APAV, either
singly (APAV-a and APAV-b) or in combination (APAV2).
TAP is a combination of APAV2 with TPR, a mutant
shown to be deficient in Hsp70 binding (20), and GAP is
an APAV2 combination with GGMP, a mutant in which Hip's
GGMP repeat region is deleted. N-303 is a previously described
truncation mutant lacking the C-terminal 66 amino acids of Hip
(20).
The ability of Hip mutants to enter PR complexes was tested in the
following manner. Plasmids encoding wtHip and mutant Hip proteins were
expressed in vitro in the presence of [35S]methionine to
produce radiolabeled products. An aliquot of each synthesis mixture was
individually added to normal rabbit RL containing an ATP-regenerating
system and the Hsp90-binding drug GA (28). GA competes for
ATP binding to Hsp90 (10, 22) and blocks formation of mature
PR complexes while enhancing recovery of early and intermediate complexes containing Hsp70, Hsp90, Hop, and Hip (27). The RL mixtures were then used to support the cell-free assembly of PR complexes with each sample containing an equimolar amount of
radiolabeled wtHip or mutant.
PR complexes were separated by SDS-PAGE and Coomassie blue stained
(Fig. 2, upper panel), and then the dried
gel was subjected to autoradiography (lower panel). Stained bands
representing recombinant chicken PR-A, nonreceptor components of the
complexes, and antibody heavy chains are indicated. The Hip bands seen
in the upper panel are endogenous rabbit Hip from RL; its abundance in
the assembly mixtures far exceeds the amount of radiolabeled human Hip
forms added to the mixtures. The unmarked band below rabbit Hip and other minor bands are RL proteins that bind nonspecifically to antibody
resins. As measured by densitometry of bands on the autoradiograph, APAV-a and APAV-b were recovered in PR complexes at approximately one-half the level of wtHip, but recovery of the combination
APAV2 mutant was only 10% of the level of wtHip.
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FIG. 2.
Association of radiolabeled APAV mutants with PR
complexes. PR complexes were assembled in vitro by using RL
supplemented with radiolabeled wtHip (wt) or the three APAV mutants.
After assembly, components were separated by SDS-PAGE and visualized by
Coomassie blue staining (upper panel). Proteins associated with
recombinant chicken PR-A (cPRA) are indicated on the left. Also labeled
are the heavy-chain bands (HC) from antibody used to immobilize PR-A
for assembly reactions. The gel was dried and autoradiographed (lower
panel) to reveal binding by radiolabeled Hip forms.
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Since the amount of radiolabeled Hip forms present in the RL assembly
mixtures is much less than the amount of endogenous Hip, no conclusions
as to whether Hip mutants disrupt PR assembly can be drawn from this
experiment. However, there is a clear deficit of DPEV mutants relative
to wtHip recovered in PR complexes.
Interactions of Hip mutants with Hsp70.
Hip transiently
associates with PR complexes (19, 25, 27) indirectly through
its binding to the ATPase domain of Hsp70 (12). Mutants were
tested for their association with Hsp70, as shown in Fig.
3. Radiolabeled Hip forms were added to
RL, and Hsp70 complexes were immunoprecipitated with anti-Hsp70
monoclonal antibody BB70. Hip binding to Hsp70 is normally salt
sensitive, but mutant N-303 was earlier observed to bind Hsp70 in a
salt-resistant manner (20). The immunoprecipitates were
divided and washed in buffer either lacking NaCl or containing 0.5 M
NaCl. Samples were separated by SDS-PAGE and Coomassie blue stained
(Fig. 3, upper panel), and the dried gel was subjected to
autoradiography (lower panel).
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FIG. 3.
Coprecipitation of Hip forms with Hsp70. Hsp70 complexes
were immunoprecipitated from RL supplemented with the radiolabeled Hip
forms indicated above the lanes (wt, wtHip). An equimolar amount of
each radiolabeled Hip form was added to separate mixtures for
immunoprecipitation reactions. Immunoprecipitates were washed in buffer
containing either no additional salt () or 0.5 M NaCl (+). Proteins
in resin complexes were separated by SDS-PAGE and visualized by
Coomassie blue staining (upper panel). Hsp70 and the coprecipitating
proteins Hop and Hsp90 are indicated on the left. Hip is not evident in
the stained gel due to its comigration with anti-Hsp70 BB70 heavy chain
(HC). Coprecipitating radiolabeled Hip forms were detected by
autoradiography of the dried gel (lower panel).
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In the stained gel, the coprecipitation of Hsp90 and Hop with Hsp70 is
evident, but endogenous rabbit Hip in Hsp70 complexes
is not apparent,
since Hip comigrates with the heavy chain of
BB70. Previous studies
have shown that Hsp90 does not directly
interact with Hsp70 but is
recovered due to the mutual binding
of Hsp70 and Hsp90 with Hop
(
4). Hip and Hop bind separate
sites on Hsp70, since either
protein binds Hsp70 alone and the
two proteins can bind Hsp70
concomitantly (
19).
As observed previously (
20), wtHip and
GGMP coprecipitate
with Hsp70 in a salt-sensitive manner,
TPR shows no binding
to
Hsp70, and N-303 binds to Hsp70 in a salt-resistant manner.
It has been
shown that Hip's TPR and adjacent highly charged region
are required
for Hsp70 binding (
15,
20). To explain the salt-resistant
binding of N-303 to Hsp70, it was previously proposed that the
mutant
is capable of binding both the ATPase and the peptide-binding
domains
of Hsp70, with the exposed GGMP in the truncated protein
being a
substrate for Hsp70's peptide-binding domain. Supporting
this view was
the observed return to salt-sensitive binding in
the truncation mutant
N-276, which lacks the GGMP repeat (
20).
If true, then
mutant N-303 would be expected to compete with misfolded
protein
substrates for binding to Hsp70. However, the proposal
for dual binding
to Hsp70 domains by N-303 must be revised in
light of the results with
APAV
2,
GAP, and
TAP. As seen in Fig.
3,
APAV
2 and
GAP, which lacks the GGMP repeat, both bind
Hsp70 in a salt-resistant
manner. On the other hand, little binding to
Hsp70 is observed
with
TAP, which retains the GGMP repeat. These
results suggest
that binding of Hip mutants to Hsp70 may be strictly
dependent
on Hip TPR interactions with Hsp70's ATPase domain, although
GGMP
and C-terminal Hip sequences can influence the biochemical nature
of Hip interactions, perhaps by altering the conformation of Hip's
TPR
domain.
Binding of wtHip and APAV
2 to the separate Hsp70 domains
was assessed directly (Fig.
4). Purified
recombinant Hip forms were
bound to a covalently coupled immunoaffinity
resin and suspended
in buffer supplemented with the radiolabeled
ATPase domain (Hsc70-AD)
or peptide-binding domain (Hsc70-PD).
After incubation, the resins
were washed in either low- or high-salt
buffer, and proteins were
separated by SDS-PAGE. Coomassie blue
staining was used to demonstrate
the quantity of wtHip or
APAV
2 in each sample. Autoradiographs of the stained gels
revealed
the salt-sensitive binding of either Hip form to Hsc70-AD.
However,
no specific interaction was detected between Hsc70-PD and
either
wtHip or APAV
2. Recovery of radiolabeled Hsc70-PD in
the Hip samples was no
greater than its recovery on immunoaffinity
resin lacking Hip.
Although Hsc70-PD binding in this assay is somewhat
promiscuous,
specific binding of Hsc70-PD to PR, a natural substrate
for Hsp70,
was detected, indicating that the isolated peptide-binding
domain
retains its ability to bind substrates. As judged by
densitometry
of bands on the autoradiograph, the association of
Hsc70-PD with
PR was 2.5-fold greater than its association with control
resin
lacking PR. As expected for Hsp70-substrate interactions, the
enhanced recovery of Hsc70-PD with PR was maintained following
washing
with 0.5 M NaCl (not shown).
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FIG. 4.
Association of wtHip and APAV2 with separate
Hsp70 domains. For the main panels, recombinant wtHip (wt) or
APAV2 was adsorbed to a covalently coupled immunoaffinity
resin. The Hip resins or resin lacking bound Hip (control) were
incubated in WB plus 5 mM ADP and 5 mM MgCl2. The buffer
was further supplemented with the radiolabeled synthesis mixtures for
the ATPase domain of Hsc70 (Hsc70-AD) or the peptide-binding domain
(Hsc70-PD). Hip immunoprecipitates were divided and washed in buffer
lacking () or containing (+) 0.5 M NaCl. As a positive control for
Hsc70-PD binding to substrate, radiolabeled Hsc70-PD was incubated with
immunoadsorbed PR or with immunoaffinity resin lacking PR (ctrl). After
SDS-PAGE separation of components, the gels were Coomassie blue stained
(Hip forms) and autoradiographed ([35S]Hsp70AD and
[35S]Hsp70-PD).
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Hip binds Hsp70, and Hsp70 can simultaneously bind Hop-Hsp90
(
19). To test whether APAV
2 alters Hsp70
interactions with Hop-Hsp90, recombinant Hip forms
were bound to
immunoaffinity resin and incubated with RL. As determined
from gel
analysis of immunoprecipitates (Fig.
5,
upper panel),
Hop and Hsp90 coprecipitate equally with wtHip and
APAV
2.
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FIG. 5.
Bulk interactions of Hip forms and their association
with an Hsp70 mutant lacking ATPase activity. Recombinant wtHip or
APAV2 was preadsorbed to a covalently coupled
immunoaffinity resin. The resins were subsequently incubated in RL
containing an ATP-regenerating system. Proteins on the resin complexes
were separated by SDS-PAGE and Coomassie blue stained (upper panel).
Stained bands representing coprecipitating Hsp70, Hop, and Hsp90 are
indicated on the left. Radiolabeled Hsp70 forms were included in the
bulk reaction mixtures, and the association of these forms with wtHip
and APAV2 was resolved by autoradiography of the stained
gel (lower panel). Either radiolabeled wild-type Hsp70 (wt) or an
ATPase-deficient point mutant (K71E) was included in the reaction
mixtures.
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Another characteristic of Hip is that it binds Hsp70 in an
ADP-dependent manner (
12,
19). To test whether
APAV
2 differs in this regard, we compared it with wtHip for
binding
to two forms of Hsp70: wild-type Hsp70, which hydrolyzes bound
ATP to ADP, and a K71E point mutant that binds ATP but lacks any
ATPase
activity (
18). Radiolabeled Hsp70 forms were included
in
samples, and autoradiography of the gel (Fig.
5, lower panel)
revealed
that neither wtHip nor APAV
2 bound K71E.
Inhibition of PR assembly by C-terminal Hip mutants.
The
transient participation of Hip in PR assembly in vitro argues for a
functional role of Hip in this process. Evidence that Hip stabilizes
Hsp70 interactions with misfolded substrates has been presented
elsewhere (12), but we have had difficulty in demonstrating
a requirement for Hip in PR assembly. Having multiple specific
monoclonal antibodies which recognize Hip (19), we have
added them to RL in various combinations in hopes of inhibiting Hip-mediated steps in PR assembly but have observed no effects (20a). Another approach has been to immunodeplete Hip from
RL prior to assembly reactions. By this method, we have estimated that
Hip's concentration in RL is approximately 1 µM (35 to 40 µg/ml),
about the same as Hsp70's molar concentration.
Immunodepletion of Hip from RL was attempted to demonstrate a
requirement for Hip in PR assembly. However, the results were
inconclusive but informative. The image in Fig.
6A is a Western-immunostained
blot
illustrating relative Hip levels in mock-depleted and depleted
RL and
in PR complexes assembled from these lysates. Densitometry
of the RL
bands indicated that greater than 90% of the total Hip
was removed,
but Hip recovery from PR complexes was reduced only
by 40%. The
compositions of the PR complexes, whether assembled
in mock- or
Hip-depleted lysate, were identical (Coomassie blue-stained
lanes not
shown). The discrepancy between Hip removal from RL
and its recovery
from PR complexes demonstrates a selective enrichment
of Hip in PR
complexes. Possibly, the small fraction of Hip remaining
in the
depleted RL is sufficient to maintain any requirement for
Hip in the
assembly process.
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FIG. 6.
Hip involvement in PR assembly. (A) RL that was mock
depleted or immunodepleted of endogenous Hip was used for assembly of
PR complexes in vitro. The resulting PR complexes (PR lanes) and the
total RL used for assembly reactions (RL lanes) were immunostained for
the presence of Hip. (B) Purified recombinant Hip forms were added to
RL at a 10-fold molar excess over endogenous Hip levels. The RL
mixtures were used for PR assembly reactions, and the resulting PR
complexes were separated by SDS-PAGE and visualized by Coomassie blue
staining. The sample in the first lane contained no added protein, and
the sample in the final lane (control) lacked PR in the assembly
reaction mixture. Proteins associated with PR (recombinant chicken PR-A
[cPRA]), along with the PR22 heavy chains (HC), are indicated on the
left.
|
|
As an alternate functional test of Hip, PR complexes were assembled in
RL supplemented with purified, recombinant Hip forms
(Fig.
6B). The
assembly mixtures contained a 10-fold molar excess
(relative to
endogenous Hip levels in RL) of wtHip, APAV
2, or
TAP.
Several changes in PR complexes are observed exclusively
for
APAV
2. First, Hsp90 is greatly reduced in PR complexes,
essentially
down to the background level of nonspecific binding; at the
same
time, Hsp70 recovery increases. Note also that APAV
2
is readily evident in PR complexes but not on a control resin
lacking
PR. Evidently, APAV
2 arrests PR assembly at an early stage
at which Hsp70 and APAV
2 bind but formation of the
intermediate complex containing Hsp90
and Hop is prevented. Since this
effect is not seen with an equal
amount of
TAP that lacks Hsp70
binding, the inhibition of Hsp90
binding to PR by APAV
2
probably relates to APAV
2 interactions with Hsp70.
As shown in Fig.
7A, APAV
2
and the truncation mutant N-303, which lacks the DPEV-containing C
terminus, both inhibit PR assembly
in a dose-dependent manner. Again,
APAV
2 inhibited Hsp90 association, with concomitant
increases in Hsp70
and APAV
2 in the receptor complex. N-303
also efficiently blocks Hsp90
binding. Half-maximal inhibition of Hsp90
binding occurs when
Hip mutants are present in three- to sixfold molar
excess over
endogenous Hip. Unlike APAV
2, N-303 does not
cause a dose-dependent increase in Hsp70, and
recovery of N-303 on PR
complexes is somewhat less than for APAV
2. N-303, which is
66 amino acids shorter than wtHip or APAV
2, comigrates with
an RL protein that binds nonspecifically to
resins; however, a modest
dose-dependent increase in N-303's association
with PR complexes can
be detected. It is not understood why APAV
2 and N-303
differ in their effects on Hsp70 accumulation in PR
complexes, but it
might relate to C-terminal sequences other than
DPEV that are absent
from N-303 and retained in APAV
2.
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FIG. 7.
Dose dependence and specificity of PR assembly
inhibition. (A) PR complexes were assembled in RL containing the
concentrations of added APAV2 or N-303 indicated above the
lanes. Protein components on the resulting resin complexes were
separated by SDS-PAGE and Coomassie stained. (B) Similar PR
reconstitutions were performed with Hip mutants replaced by the
concentrations of the Hsp70 substrate -casein or RCMLA indicated
above the lanes.
|
|
Since Hsp70 is required at an early stage for assembly of receptor
complexes (
14,
26), anything that inhibits Hsp70 function
would be expected to inhibit PR assembly at some level. If
APAV
2 is misfolded, it could competitively inhibit Hsp70
binding to
PR. Inconsistently with this interpretation,
APAV
2 is recovered in PR complexes and Hsp70 recovery
increases. Also,
APAV
2 does not appear to bind Hsp70's
peptide-binding domain (Fig.
3 and
4). To further minimize the
likelihood that APAV
2 is competitively inhibiting Hsp70 by
binding its peptide-binding
site, we compared two frequently studied
Hsp70 substrates for
their abilities to inhibit PR assembly (Fig.
7B).
Since Hsp70
is required for PR assembly and recognizes PR as a
substrate,
other Hsp70 substrates should inhibit PR assembly to some
degree.
However, little inhibition of PR assembly was observed with a
32 µM concentration of
-casein, although inhibition by this
substrate
was observed at much higher concentrations (results not
shown).
Partial inhibition of PR assembly was observed with RCMLA, but
RCMLA is less effective as an inhibitor than either APAV
2
or N-303.
Hip is able to oligomerize (
12), and this activity maps
within the first 14 amino acids at Hip's N terminus (
20).
The
physiological significance of Hip oligomerization is unknown,
but
oligomerization-deficient N-terminal truncation mutants were
observed
to bind Hsp70 and assemble in PR complexes, similarly
to wtHip
(
20). To test if oligomerization is necessary for the
inhibitory property of APAV
2, this mutant was combined with
a truncation of Hip's first 14
amino acids. However, the monomeric
combined mutant inhibited
PR assembly, similarly to APAV
2
(results not shown).
Effects of Hip forms on Hsp70 binding dynamics.
Since Hip can
stabilize Hsp70 binding to some substrates (12), we tested
whether APAV2 or N-303 inhibits PR assembly by altering the
dynamics of Hsp70-PR interactions (Fig.
8A and B). PR assembly proceeded through
an initial pulse phase during which radiolabeled Hsp70 was present and
steady-state assembly dynamics were established. In a subsequent chase
phase, radiolabeled Hsp70 was excluded, and assembly was allowed to
proceed for different times. During both phases, a 10-fold molar excess
of recombinant wtHip, APAV2, or N-303 was present. Neither
APAV2 (Fig. 8A) nor N-303 (Fig. 8B) altered the exchange of
Hsp70 on PR complexes compared with wtHip. In all cases, the half-time to reach new steady-state levels of radiolabeled Hsp70 in PR complexes was approximately 2 min.
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FIG. 8.
Effects of mutant Hip forms on the dynamics of Hsp70
interactions. The rates of exchange of radiolabeled Hsp70 on PR
complexes (A and B) and on Hip complexes (C and D) were compared in the
presence of wtHip (A to D), APAV2 (A and C), or N-303 (B
and D). As detailed in Materials and Methods, PR or Hip complexes were
assembled in RL containing radiolabeled Hsp70 and then transferred to
fresh RL lacking radioactivity. At the times indicated, aliquots were
removed from the secondary assemblies. Each sample was separated by
SDS-PAGE, Coomassie blue stained to monitor total protein levels, and
autoradiographed to measure the binding of radioactive Hsp70. Bands
were quantitated by densitometry, and radioactive bands were normalized
to the amount of Coomassie blue-stained Hsp70 in each sample. The
resulting values are plotted as a percentage of radiolabeled Hsp70
present at initiation of the secondary assembly reactions (0 min).
|
|
In a similar set of reactions, the exchange of Hsp70 on Hip forms was
measured (Fig.
8C and D). APAV
2 and wtHip, as intein/CBD
fusion proteins, were adsorbed to chitin
resin, and N-terminally
His-tagged N-303 and wtHip were adsorbed
to Ni
2+-resin.
Negative-control samples, with
TAP-intein/CBD bound to
chitin resin
or Ni
2+-resin lacking any His-tagged protein, failed to
show any binding
by radiolabeled Hsp70. Contrasting with the similarity
of Hsp70-PR
exchange rates, the rate of Hsp70 exchange on wtHip was
markedly
lowered by mutations in Hip's C-terminal region. The
half-time
to reach new steady-state levels of radiolabeled Hsp70 on
wtHip
was approximately 1 to 2 min but was closer to 5 min with either
APAV
2 (Fig.
8C) or N-303 (Fig.
8D). Briefly, Hip mutants
differ from
wtHip in their interactions with Hsp70, but the more stable
binding
of mutant Hip to Hsp70 does not alter the normal binding and
release
of Hsp70 from PR complexes.
Hsp70 binding to Hop is inhibited by misfolded substrates.
Hip
mutants APAV2 and N-303 do not prevent binding of Hsp70 and
the mutant to PR, but PR assembly is arrested immediately prior to
incorporation of Hop and Hsp90 into intermediate complexes. Hop-Hsp90
is able to bind Hsp70-APAV2 in the absence of PR (Fig. 5)
but not, apparently, in the presence of PR. Perhaps, then, there is a
difference in Hop binding to Hsp70 that is dependent on whether Hsp70
is associated with a substrate. This was confirmed when the binding of
Hop to Hsp70 in the presence and absence of misfolded substrates was
compared (Fig. 9). Hsp70 alone or in the
presence of native BSA will readily bind immunoaffinity-purified Hop.
On the other hand, if Hsp70 is preincubated with the substrate
-casein or RCMLA, binding of Hsp70 to Hop is reduced to the
background level observed with immunoaffinity resin alone.
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FIG. 9.
Inhibition of Hop binding to Hsp70 in the presence of
misfolded substrates. Hop was immunoadsorbed from RL in the presence of
0.5 M NaCl to dissociate Hsp70 and Hsp90. Hop-resin (lane Hop) was then
incubated with purified Hsp70 (lane Hsp70) or with Hsp70 preincubated
with a 10-fold molar excess of -casein, RCMLA, or BSA as indicated
above the lanes. In the final lane, immunoaffinity resin lacking Hop
was incubated with Hsp70. Migration positions for Hsp70, Hop, and the
anti-Hop immunoglobulin heavy chain (HC) are indicated on the left.
|
|
| |
DISCUSSION |
The normal assembly pathway for PR complexes (25, 27)
is illustrated in Fig. 10A. PR must
reach the mature complex in order to bind hormone with efficiency and
high affinity, and since the mature complex is not inherently stable,
complexes must be constantly reassembled to maintain a PR pool
competent for hormone-dependent activation.
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FIG. 10.
(A) Normal assembly cycle. During cell-free assembly of
PR complexes, an ordered pathway is followed. (1) Hsp70 and Hip bind at
an early stage. (2) An intermediate complex containing Hsp70, Hop, and
Hsp90 is formed. (3) The functionally mature complex contains Hsp90,
p23, and any one of three immunophilins (Imph.). Hormone binding by PR
is deficient (dashed outline of PR) until the mature complex is formed.
The mature complex is not stable; if PR dissociates without binding
hormone, it reenters the assembly cycle. (B) APAV2-arrested
assembly. Hip mutant APAV2, or N-303, is permissive for
Hsp70 binding and dissociation but prevents the coassociation of
Hop-Hsp90 and Hsp70 on PR. (C) Hip-mediated transition in PR complexes.
It is proposed that Hip normally facilitates binding of Hop to Hsp70
when Hsp70 is associated with PR, thus promoting progression of PR
assembly to the intermediate complex containing Hsp90. The indirect
association of Hsp90 with PR complexes may favor displacement of Hsp70
and direct binding of Hsp90 to PR.
|
|
Whereas the compositions of receptor complexes at different assembly
stages are fairly well defined, the mechanisms for transition from one
complex to the next are not. It is known that blocking of p23 function
prevents the transition from intermediate to mature complexes (13,
16, 27), inhibition of Hop function blocks formation of
intermediate complexes (4-6), and inhibition of Hsp70
prevents all assembly steps (14, 26). What we've found here
is that certain Hip mutants, while allowing Hsp70 to transiently associate with PR, prevent formation of intermediate complexes containing Hop and Hsp90 (Fig. 10B).
The initial characterization of APAV2 showed that the
radiolabeled protein was underrepresented in PR complexes (Fig. 2), yet the experiments whose results are presented in Fig. 6B and 7A show
enhanced recovery of APAV2 in PR complexes. These seemingly
paradoxical results can be explained in the following manner. When
APAV2 is present in RL at low levels relative to endogenous
Hip, as in Fig. 2, assembly can proceed via the actions of endogenous Hip. The small pool of PR complexes containing APAV2 do not
progress to transitional stages and quickly dissociate. In contrast,
when APAV2 is the dominant Hip form (as in Fig. 6B and 7A),
assembly progression is efficiently blocked, and the pool of
PR-Hsp70-APAV2 complexes increases even though the
complexes remain dynamic.
Mechanism for inhibition of PR assembly by Hip mutants.
Inhibition of PR assembly by C-terminal Hip mutants requires binding of
the Hip mutants to Hsp70 (Fig. 6B, lane TAP), and the mutants retain
several of wtHip's Hsp70-binding characteristics. For example, the
mutants still bind the ATPase domain of Hsp70 in an ADP-dependent
manner (Fig. 3 to 5). Also, the Hip mutants do not block Hsp70 binding
to PR (Fig. 6B and 7), nor do they alter the dynamics of Hsp70-PR
interactions (Fig. 8A and B). One distinguishing characteristic of the
Hip mutants is their more stable binding to Hsp70. Unlike wtHip,
association of APAV2 and N-303 with full-length Hsp70 is
resistant to increased ionic strength (Fig. 3), and the mutants
exchange on Hsp70 more slowly than wtHip (Fig. 8C and D). More stable
binding of Hip mutants to Hsp70 competitively favors their occupation
of Hsp70, thus accounting for the dominance over wtHip of mutant Hip
interactions with Hsp70. However, more stable Hsp70 binding alone
probably does not account for inhibition of PR assembly.
The sequences mutated in APAV
2 and N-303 are similar to
sequences in the C-terminal region of Hop. Also, Hip and Hop both bind
Hsp70 and can do so concomitantly. Hop is required for the entry
of
Hsp90 into steroid receptor complexes (
4-6), and the Hip
mutants
tested in this study arrest PR assembly prior to incorporation
of Hop into PR complexes. Hence, there is a potential connection
between Hip function and Hop's ability to enter PR complexes.
While Hop can bind substrate-free Hsp70 alone (Fig.
9) or in the
presence of APAV
2 (Fig.
5), Hop is unable to associate with
Hsp70 when misfolded
substrates are present (Fig.
9). This suggests
either that Hop
is sterically excluded from binding Hsp70 by misfolded
substrates
or that the conformation of Hsp70 recognized by Hop is
altered
by substrate binding. We have been unsuccessful in mapping
Hop's
binding site on Hsp70 using Hsc70-AD, Hsc70-PD, or a number of
additional Hsp70 truncation mutants (
4b), indicating that
Hop's
binding is sensitive to Hsp70's overall conformation. This is
further suggested by Hop's failure to recognize the ATP-bound
form of
Hsp70. Finally, Hop can clearly associate with Hsp70 in
PR
complexes
indeed, Hop is required for progression of PR
assembly
arguing
against a strict steric exclusion of Hop from this
particular
Hsp70-substrate complex. It seems most likely that the
occupancy
status of Hsp70's peptide-binding domain conformationally
alters
the binding site for Hop, requiring some mechanism to facilitate
Hop binding to PR-associated Hsp70, Hip may be serving this role.
A model (Fig.
10C) in which Hip and Hop direct the transition from
early PR complexes (containing Hsp70 and Hip) to intermediate
complexes
(containing Hsp70, Hop, and Hsp90) is proposed. In the
scheme shown,
Hsp70 binds to PR, apparently recognizing PR as
a misfolded protein;
Hip then binds Hsp70 and induces a conformational
change that
facilitates Hop-Hsp90 binding. Hop binding may promote
release of Hip
from Hsp70, or Hip dissociation may occur spontaneously.
The
Hop-mediated localization of Hsp90 to intermediate complexes
might
favor direct binding of Hsp90 to PR with concomitant displacement
of
Hsp70 as a precedent to formation of mature complexes.
It is clear that the two domains of Hsp70 are conformationally linked
and mutually influenced by the presence of ATP or ADP
in the ATPase
domain and by the presence or absence of a substrate
in the
peptide-binding domain (
2,
7,
9). The influence
of the
peptide domain on ATPase domain interactions with other
proteins is
evident in two examples presented here. First, mutant
APAV
2
binds Hsp70's isolated ATPase domain in a salt-sensitive manner,
but
APAV
2 binding is salt resistant with full-length Hsp70
(Fig.
3 and
4). We found no direct interaction between
APAV
2 and Hsp70's peptide-binding domain to account for
salt-resistant
binding. Second, Hop readily binds free Hsp70 but fails
to bind
Hsp70 in the presence of misfolded substrates (Fig.
9).
However,
under the right conditions (namely, the presence of wtHip),
Hop
can bind Hsp70 while its peptide-binding domain is occupied with
PR.
Recently, we have generated mutations in the DPEV region of Hop and
found that the mutants fail to bind Hsp70 and fail to
support PR
assembly (
4a). This observation strengthens the potential
importance of Hip's and Hop's DPEV sequences in directing assembly
of
functionally mature PR complexes. In conclusion, Hip may have
evolved
as a coadapter with Hop to enhance and coordinate recognition
of
steroid receptors, and perhaps other Hsp70 substrates, by Hsp90.
| |
ACKNOWLEDGMENT |
This work was supported by NIH grant R01-44923.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pharmacology, University of Nebraska Medical Center, Omaha, NE
68198-6260. Phone: (402) 559-8604. Fax: (402) 559-7495. E-mail:
dfsmith{at}mail.unmc.edu.
| |
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Abstract
Introduction
