1 Turku Centre for Biotechnology, University of Turku, Åbo Akademi
University, BioCity, PO Box 123, FIN-20521 Turku, Finland
2 Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku,
Finland
3 Department of Biology, Åbo Akademi University, Turku, Finland
Author for correspondence (e-mail:
lea.sistonen{at}btk.utu.fi)
Accepted 16 May 2003
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Summary |
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Key words: Heat-shock response, Hsf, Hsp70, Stress granule
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Introduction |
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HSF1 is the most studied heat-shock transcription factor and is the
classical HSF, which responds to elevated temperatures and other forms of
protein damaging stress (Pirkkala et al.,
2001). In addition, HSF1 has been shown to be involved in
extra-embryonic development and female fertility in mice
(Xiao et al., 1999
;
Christians et al., 2000
). Upon
stress, HSF1 is rapidly converted from a monomer to a trimer, is inducibly
phosphorylated and concentrated in the nucleus to activate heat-shock gene
transcription (Baler et al.,
1993
; Rabindran et al.,
1993
; Sarge et al.,
1993
). In addition to HSF1, chicken HSF3 is activated by similar
but more severe stressors (Nakai and
Morimoto, 1993
; Tanabe et al.,
1997
). In HSF3-deficient cells the formation of HSF1 trimers is
hampered and Hsp expression is reduced upon heat shock
(Tanabe et al., 1998
). The
interdependency between HSF1 and HSF3 in avian cells is so far the only
example of cross-talk between different HSFs. However, no physical interaction
has been reported between these two proteins.
A characteristic feature of cellular stress in human cells is the
organization of HSF1 into specific subnuclear structures, termed stress
granules. These irregularly shaped granules have been described to form under
various stress conditions, including exposure to heat, cadmium, azetidine and
proteasome inhibitors (Cotto et al.,
1997; Jolly et al.,
1997
; Jolly et al.,
1999
; Holmberg et al.,
2000
). Stress-induced HSF1 granules have been found in all
investigated primary and transformed human cells but not in rodent cells
(Sarge et al., 1993
;
Mivechi et al., 1994
;
Cotto et al., 1997
;
Jolly et al., 1999
). The
appearance of stress granules correlates positively with the inducible
phosphorylation and transcriptional activity of HSF1
(Sarge et al., 1993
;
Cotto et al., 1997
;
Jolly et al., 1999
;
Holmberg et al., 2000
).
Furthermore, HSF1 dissociates from the stress granules and relocalizes
diffusely in the cell during attenuation and recovery from stress; upon
subsequent exposure to stress, the granules reappear at the same sites
(Jolly et al., 1999
). The
stress granules have not been shown to represent other previously described
nuclear structures (Cotto et al.,
1997
). In addition to HSF1, several RNA binding proteins such as
heterogeneous nuclear ribonucleoprotein (hnRNP) HAP (hnRNP A1 interacting
protein), hnRNP M, Src-activated during mitosis (Sam68) and certain SR
(serinearginine) splicing factors have been identified in stress granules
(Weighardt et al., 1999
;
Denegri et al., 2001
).
Recently, stress granules were revealed to associate with human chromosome
9q11-q12, corresponding to a large block of heterochromatin composed primarily
of satellite III repeats adjacent to the centromere
(Jolly et al., 2002
).
Furthermore, Denegri et al. (Denegri et
al., 2002
) have reported that, in addition to chromosome 9,
chromosomes 12 and 15 also contain nucleation sites for stress granules. The
functional significance of stress granules is still unknown.
The autoregulation of the heat-shock response is facilitated by a direct
interaction between HSF1 and molecular chaperones, such as Hsp70 and Hsp90
with their cochaperones. This has been established in several biochemical,
genetic and cell physiological studies
(Abravaya et al., 1992;
Baler et al., 1992
;
Kim et al., 1995
;
Ali et al., 1998
;
Shi et al., 1998
;
Zou et al., 1998
;
Bharadwaj et al., 1999
;
Morimoto, 2002
). Under normal
physiological conditions, HSF1 exists in a repressed state associated with
molecular chaperones. Upon stress, it is rapidly released from the complex,
trimerized and hyperphosphorylated, and acquires DNA-binding and
transcriptional activity. When exposed to continuous moderate heat stress, the
equilibrium is shifted back to the monomeric, dephosphorylated and Hsp-bound
state. The downregulation of HSF1 activity during prolonged stress is referred
to as attenuation of the heat-shock response, and leads to the suppression of
HSF1 reactivation capacity upon subsequent stress. A similar repression of
HSF1 activity is observed when cells recover from stressful conditions. This
acquired adaptation of cells to stress stimuli is called thermotolerance
(Morimoto, 2002
).
Biochemical characterization of HSF2 has revealed that, unlike HSF1, which
undergoes a monomer-to-trimer transition, HSF2 is mainly converted from a
dimer to a trimer upon activation, and regulation of HSF2 appears not to
include phosphorylation (Sarge et al.,
1993; Sistonen et al.,
1994
). The regulation of HSF1 and HSF2 expression varies
dramatically. In contrast to stably and constitutively expressed HSF1, the
HSF2 expression levels are regulated both transcriptionally and by mRNA
stabilization (Pirkkala et al.,
1999
). Rapid accumulation of HSF2 protein by downregulation of the
ubiquitin proteolytic pathway has provided evidence that HSF2 is a labile
protein (Mathew et al., 1998
;
Pirkkala et al., 2000
). The
regulation of HSF2 has been linked to certain development- and
differentiation-related processes, such as gametogenesis and pre- and
postimplantation development of mouse embryos
(Mezger et al., 1994
;
Sarge et al., 1994
;
Rallu et al., 1997
;
Alastalo et al., 1998
;
Eriksson et al., 2000
).
Furthermore, the human HSF2 DNA-binding activity is induced during
hemin-mediated erythroid differentiation of K562 cells
(Sistonen et al., 1992
). A
recent study by Kallio et al. (Kallio et
al., 2002
) revealed that the disruption of mouse hsf2
gene leads to brain abnormalities and defects in gametogenesis in both
genders. No correlation between HSF2 expression and activity with Hsp
expression has been obtained during differentiation-related processes,
indicating existence of other HSF2 target genes
(Rallu et al., 1997
;
Alastalo et al., 1998
;
Kallio et al., 2002
).
A few reports have proposed that HSF2 is involved in the regulation of the
stress response. According to Sheldon and Kingston
(Sheldon and Kingston, 1993),
HSF2 localizes in the nucleus and in granule-type structures in HeLa cells
exposed to heat stress. Furthermore, Mathew et al.
(Mathew et al., 2001
) have
reported that, in murine fibroblasts, HSF2 has the biochemical properties of a
temperature-sensitive protein because, upon heat shock, HSF2 is localized to
the perinuclear region, its solubility is decreased and DNA-binding activity
is diminished. This intriguing difference between human and murine cells has
also been observed with HSF1, because the stress-induced subnuclear
compartmentalization of HSF1 has been detected only in human cells
(Sarge et al., 1993
;
Denegri et al., 2002
).
Altogether, direct evidence of HSF2, human or murine, being a physiological
transcriptional regulator of heat shock genes is missing.
Despite the characterization of several biochemical properties of HSF1 and HSF2, the actual stress sensors and the signaling pathways regulating these two factors have remained obscure. In this study, we provide evidence that HSF1 and HSF2 form a physically interacting complex and co-localize in the dynamically regulated nuclear stress granules, suggesting a novel role for HSF2. We also show data suggesting that the association of HSF1 and HSF2 with stress granules might be regulated by Hsp70, and that the association is severely impaired in thermotolerant cells. Taken together, our results suggest that the regulation of the subcellular distribution of Hsp70 contributes to the regulation of HSF1-mediated heat-shock responses.
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Materials and Methods |
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Construction of plasmids and a stable cell line
Human HSF1 with C-terminal Myc tags was constructed by PCR and cloned into
EcoRI and HindIII sites in the pcDNA3.1(-)MycHis A vector
(Invitrogen) in frame with the MycHis tag
(Holmberg et al., 2001). Mouse
HSF1 with N-terminal Flag tags was a kind gift from R. I. Morimoto
(Northwestern University, IL, USA). Mouse HSF2-
and HSF2-ß with
N-terminal Flag tags have been described previously
(Pirkkala et al., 2000
).
Expression vectors encoding mouse HSF2-
and HSF2-ß with C-terminal
Myc tags were constructed by PCR and cloned into the EcoRI site in
the pcDNA3.1(-) MycHis B vector (Invitrogen) in frame with the MycHis tag. The
mouse HSF2-ß deletion mutants (Fig.
4A) were constructed by PCR and cloned into the EcoRI and
EcoRV sites in frame with the N-terminal Flag tag in pFLAG-CMV-2
(Kodak). All PCR-amplified products were sequenced to exclude the possibility
of second site mutagenesis. HSF1-Myc-His stably overexpressing cell line (4H9)
was generated by electroporating 30 µg of HSF1-Myc-His to K562 cells as
described below. After 2 days of recovery, neomycin-resistant cells were
selected by growing the cells in medium containing 500 µg ml-1
G418 (Life Technologies) for 2 weeks. Neomycin-resistant single cell clones
were picked out after serial dilutions and screened for HSF1-Myc-His
expression by western blotting. Cells were routinely grown in the presence of
500 µg ml-1 G418.
|
Transfections
K562 and HeLa cells were transfected by electroporation (975 µF, 220 V)
using a Bio-Rad Gene Pulser electroporator. For this procedure,
5x106 cells were washed, resuspended in 0.4 ml of Optimem
(Gibco-BRL) and placed in a 0.4-cm-gap electroporation cuvette (BTX). Plasmid
DNA (30 µg) was added and, after a brief incubation at room temperature,
the cells were subjected to a single electric pulse. Thereafter, cells were
cultured at 37°C for 40 hours prior to the indicated experimental
treatments.
Indirect immunofluorescence and confocal microscopy
For immunofluorescence analysis, HeLa cells growing on coverslips were
washed with PBS and simultaneously fixed and permeabilized for 15 minutes in
0.5% Tween 20 in PBS containing 3% paraformaldehyde, or the cells were fixed
for 15 minutes in 4°C methanol-acetone (1:1). After three washes with PBS,
cells were incubated for 1 hour with blocking solution (20% boiled normal goat
serum in PBS). Rabbit anti-HSF1 (Holmberg
et al., 2000), rat anti-HSF1 (Neomarkers), rabbit anti-HSF2
(Sarge et al., 1993
), mouse
anti-Hsp70 (SPA-810; StressGen), mouse anti-Flag M2 (Sigma) or mouse anti-Myc
(Sigma) antibody (1:500 dilutions) were added for 1 hour. After washes with
PBS, the bound primary antibodies were detected using goat anti-mouse
antibodies (1:400 dilution for 1 hour; Alexa 488, Molecular Probes),
Cy3-conjugated donkey anti-rabbit antibodies (1:400 dilution for 1 hour;
Jackson ImmunoResearch Laboratories), goat anti-rat antibodies (1:400 dilution
for 1 hour; Alexa 568, Molecular Probes) or donkey anti-rabbit antibodies
(1:400 dilution for 1 hour; Alexa 488, Molecular Probes). The DNA was stained
with DAPI (4',6-diamidino-2-phenylindol, Sigma) for 2 minutes before
final washes and mounting with Vectashield (Vector Laboratories). The cells
were analyzed and photographed using a Leica DMR fluorescence microscope
equipped with a digital Hamamatsu ORCA CCD camera or Leica TCS40 confocal
laser scanning microscope using the SCANware 4.2a program. Images were further
processed using Adobe Photoshop and CorelDraw software.
Immunoprecipitation and immunoblotting assays
For in vivo coimmunoprecipitation experiments, transiently transfected
cells were lysed in 200 µl of lysis buffer [25 mM HEPES, 100 mM NaCl, 5 mM
EDTA, 0.5% Triton X-100, 20 mM ß-glycerophosphate, 20 mM
para-nitrophenyl phosphate, 100 µM ortovanadate, 0.5 mM
phenylmethylsulfonyl fluoride, 1 mM dithiotreitol, 1x complete mini
protease inhibitor cocktail (Roche Diagnostics)] supplemented with 20 mM
N-ethylmaleimide, followed by centrifugation for 25 minutes at 15,000
g at 4°C. After protein extraction, 200-500 µg total
cell protein was preincubated with slurry of protein-G/Sepharose (Amersham
Pharmacia Biotech) in TEG buffer (20 mM Tris-HCl pH 7.5, 1 mM EDTA, 10%
glycerol) containing 150 mM NaCl and 0.1% Triton X-100 for 30 minutes at
4°C followed by a brief centrifugation. The precleared cellular lysate was
incubated with anti-HSF1 (Neomarkers), anti-HSF2 (Neomarkers), anti-Flag or
anti-Myc antibodies at room temperature for 30 minutes under rotation, after
which 40 µl of a 50% slurry of protein-G/Sepharose was added to the
reaction mixture and incubated for 12 hours at 4°C under rotation. After
centrifugation, the Sepharose beads were washed with supplemented TEG buffer
and the immunoprecipitated proteins were run on 8% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose filter (Protran
Nitrocellulose; Schleicher & Schuell) for immunoblotting. For detection of
protein input and expression levels, 12 µg of protein was analyzed by
SDS-PAGE as described above. HSF1 was detected by polyclonal antibodies
specific to mouse and human HSF1 (Sarge et
al., 1993; Holmberg et al.,
2000
), HSF2 by polyclonal antibodies specific to mouse HSF2
(Sarge et al., 1993
), the
inducible form of Hsp70 by 4g4 (Affinity Bioreagents), Hsc70 by SPA-815
(StressGen). Horseradish peroxidase (HRP)-conjugated secondary antibodies were
purchased from Promega and Amersham. The blots were developed with an enhanced
chemiluminescence method (ECL; Amersham Pharmacia Biotech).
Cells (5x106 each of 4H9 and K562) were labeled with 0.25
mCi ml-1 of TRAN35S Label (ICN) for 16 hours. Cells were lysed in 1
ml of modified RIPA buffer (50 mM TRIS pH 7.4, 150 mM NaCl, 1% NP-40, 0.1%
deoxycholate, 1 mM EDTA, 100 µM orthovanadate, 2 mM NaF) and the
immunoprecipitation with anti-c-Myc (Sigma) antibodies was performed as above.
Immunoprecipitated proteins were detected from the gels by autoradiography. To
identify the bands, 1x109 4H9 and K562 cells were lysed and
immunoprecipitated as above. The gels were silver stained
(O'Connell and Stults, 1997)
and the bands were identified using standard matrix-assisted laser desorption
ionization mass spectrometry.
Gel mobility shift assay
Whole cell extracts were prepared from experimentally treated cells as
previously described (Mosser et al.,
1988) and incubated (12 µg protein) with a
32P-labeled oligonucleotide representing the proximal HSE of the
human hsp70 promoter. The protein-DNA complexes were analyzed on a
native 4% polyacrylamide gel as described previously
(Mosser et al., 1988
).
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Results |
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|
Co-localization of HSF1 and HSF2 in the nuclear stress granules
The heat-induced localization of HSF1 and HSF2 in granules did not occur
randomly, but a co-localization of these two endogenous factors was detected
after a 1-hour heat shock, as shown by double staining
(Fig. 2A). To ensure that
finding the HSF2 stress granules was not a result of cross-reactivity between
the antibodies against HSF1 and HSF2, we constructed both N- and C-terminally
Flag- and Myc-tagged HSF1 and HSF2 plasmids. Upon transient expression in HeLa
cells, the subcellular localization of ectopic HSF1 and HSF2 was analyzed.
Immunofluorescence analyses with monoclonal antibodies against Flag and Myc
epitopes showed a similar nuclear pattern
(Fig. 2B) to the experiments
conducted with the antibodies against HSF1 and HSF2
(Fig. 1A,B,
Fig. 2A). The co-localization
between HSF1 and HSF2 was further confirmed by confocal microscopy (data not
shown). Double-staining experiments were also performed as in
Fig. 1B to study the
co-localization of HSF1 and HSF2 at different phases of granule development.
In these studies, co-localization of HSF1 and HSF2 was observed from the
compartmentalization to the dissociation of these factors from stress granules
(data not shown). It is worth noticing that, although there was prominent
co-localization of HSF1 and HSF2 in the same subnuclear structures, some
heterogeneity occurred (i.e. stress granules were detected in which HSF1 but
not HSF2 was present).
|
Dynamic interaction between HSF1 and HSF2
The dynamic localization of HSF1 with HSF2 in stress granules upon heat
shock provided new evidence for HSF2 functioning as a heat-responsive factor,
and for the stress-induced regulation of HSF1 and HSF2 being closely related.
This finding prompted us to investigate whether a similar heat-induced
subnuclear distribution of HSF1 and HSF2 could be caused by their physical
interaction. Myc-tagged HSF1 was transiently or stably expressed in K562 cells
and the endogenous HSF2 was coimmunoprecipitated with both transiently
(Fig. 3A, left) and stably
(Fig. 3A, right) overexpressed
HSF1. Furthermore, monoclonal anti-HSF2 antibodies were used to
immunoprecipitate the endogenous HSF2, and western blotting showed that the
overexpressed HSF1 coimmunoprecipitated with HSF2
(Fig. 3B).
|
To analyze the dynamics of the interaction during the course of heat-shock
response and recovery periods, endogenous HSF1 from HeLa cells was
immunoprecipitated with monoclonal anti-HSF1 antibody. The immunoprecipitated
complexes were analyzed by western blotting, and dynamic interaction of HSF1
and HSF2- and -ß isoforms
(Pirkkala et al., 2001
) was
observed at different time points (Fig.
3C). At control temperature and heat shock up to 30 minutes,
HSF2-ß was the major HSF2 isoform in the complex, whereas the amount of
HSF2-
began to increase from 30 minutes to 1 hour and similar amounts
of both isoforms were detected in the complex. After 1 hour of heat treatment,
the levels of HSF2-ß in the complex rapidly declined and, at 2-4 hours of
continuous heat shock, HSF2-
was the major HSF2 isoform interacting
with HSF1. Upon prolonged heat shock (>4 hours) and recovery, the amount of
HSF2-ß began to increase, restoring the ratio of the HSF2 isoforms
observed at control temperature (Fig.
3C, Fig. 8B). The
changes in the interaction stoichiometry between HSF1 and HSF2 isoforms, as
shown in immunoprecipitation samples (Fig.
3C, top), were reflected also in western blotting of the lysates
(Fig. 3C, bottom), indicating
altered solubility of the HSF2 isoforms during different phases of the
continuous heat shock.
|
After 3 hours of recovery from a 1-hour heat shock, when cells acquire thermotolerance, the interaction between HSF1 and HSF2 was downregulated (Fig. 3C, Fig. 8B). In contrast to the dynamic regulation of HSF2 isoforms detected both in the lysates and protein complex, the downregulation of HSF1-HSF2 interaction seen after recovery from stress cannot be explained by changes in solubility. This suggests that there are modifications in the HSF-associated protein complex during thermotolerance and further confirms the dynamic nature of HSF1-HSF2 interaction.
Oligomerization domain is essential for the interaction between HSF1
and HSF2
Different members of the HSF family share a similar structure, consisting
of a leucine-rich oligomerization domain (HR-A/B) adjacent to the N-terminal
DNA-binding domain, and another leucine-rich region near the C-terminus (HR-C)
(Rabindran et al., 1991;
Sarge et al., 1991
;
Schuetz et al., 1991
). To
examine whether the interaction of HSF1 and HSF2 depended on an intact
oligomerization domain, we constructed a series of Flag-tagged HSF2 deletion
mutants (Fig. 4A). The
Myc-tagged HSF1 was transiently expressed with these HSF2 mutants in K562
cells, and HSF1 was immunoprecipitated with anti-Myc antibody, and the
presence of HSF2 in the complex was analyzed. As shown in
Fig. 4B, HSF2 mutants lacking
HR-A/B (HSF2 203-517 and HSF2
126-201) did not form complexes with
HSF1.
HSF2 is not found in the protein complex binding to the HSE upon heat
shock
Considering that HSF2 interacts with HSF1, we wanted to investigate whether
the HSF1-HSF2 heterocomplex could bind in vitro to the HSE of human hsp70
promoter. Flag-tagged HSF1 and Myc-tagged HSF2 were transiently expressed in
K562 cells and a strong heat-induced DNA-binding activity was detected in the
lysates (Fig. 5A, left). The
spontaneous DNA-binding activity in untreated samples was due to
overproduction of HSFs. To analyze the DNA-binding complexes, we performed
antibody perturbation assays with anti-Flag and anti-Myc antibodies to detect
the presence of HSF1 or HSF2 molecules, respectively. As shown in
Fig. 5A (right), the anti-Flag
(HSF1) antibody supershifted the heat-induced DNA-binding complexes
completely, whereas the anti-Myc (HSF2) antibody had no effect on the complex.
Because hemin induces the DNA-binding activity of HSF2
(Pirkkala et al., 1999), a
hemin-treated sample expressing Myc-tagged HSF2 was included as a positive
control for HSF2 DNA-binding activity and anti-Myc antibody. The existence of
HSF1-HSF2 complex in the same lysates was determined by coimmunoprecipitation
(Fig. 5B).
|
To exclude the possibility that the C-terminal Myc-epitope in the HSF2 fusion protein was being masked in the heat-induced DNA-binding complex and could therefore not be detected, we repeated the above mentioned experiments using C-terminally Myc-tagged HSF1 and N-terminally Flag-tagged HSF2. The results were identical to those in Fig. 5, with only HSF1 being detected in the HSE-binding complex (data not shown). These results were also confirmed by overexpressing HSF2 alone, and no HSF2 was detected in the heat-induced HSE-binding complex (data not shown).
HSF2 influences the localization of HSF1 in nuclear stress
granules
Based on the obtained results, the HSF1-HSF2 complex formation could have
significance in other steps of the stress-regulated signaling pathways
unrelated directly to the HSE-binding and heat-shock gene expression. The
appearance of stress-induced nuclear granules has been shown to coincide with
HSF1 activation (Cotto et al.,
1997; Jolly et al.,
1999
; Holmberg et al.,
2000
), so we investigated whether the HSF1-HSF2 interaction
affects the subnuclear compartmentalization of HSF1. For this purpose, the
Flag-tagged HSF2 deletion mutants (Fig.
3A) were transiently transfected into HeLa cells and double
staining was performed with anti-Flag and anti-HSF1 antibodies. As shown in
Fig. 6A, the full-length HSF2
co-localized with HSF1 during heat shock. Unexpectedly, the DNA-binding-domain
deletion mutant (HSF2 108-517), which was able to interact physically with
HSF1 (Fig. 4B), did not
localize in the granules, and also the translocation of HSF1 into the granules
was severely impaired (Figs.
6A,6D).
The HR-A/B oligomerization domain deletion mutants (HSF2 203-517 and HSF2
126-201) could not interact with HSF1
(Fig. 4B) but did not
translocate into the granules; instead, these cells displayed normal
localization of HSF1 in the granules (Figs.
6B,6D).
The C-terminal deletion mutant (HSF2 1-391), which was capable of interacting
with HSF1 (Fig. 4B), localized
spontaneously to granules and, intriguingly, HSF1 showed clear co-localization
with this HSF2 mutant at both control and elevated temperatures (Figs.
6C,6D).
Taken together, the immunofluorescence analyses, combined with the
coimmunoprecipitation results (Fig.
4B), indicate that HSF2 could affect the translocation of HSF1
into the nuclear stress granules, because either induction or prevention of
HSF1 subnuclear compartmentalization was observed depending on which HSF2
mutant was used.
|
|
Localization of HSF1 and HSF2 in stress granules is suppressed in
thermotolerant cells
In addition to co-localization of HSF1 and HSF2 in the same dynamically
regulated protein complex, we observed profound changes in subnuclear
compartmentalization and HSF1-HSF2 interaction during attenuation and recovery
of the heat-shock response. To address the question of how thermotolerance
affects the localization of HSF1 and HSF2 in stress granules, we induced
thermotolerance by exposing HeLa cells to a 1-hour heat shock, after which the
cells were allowed to recover at normal temperature for 3 hours. During
recovery, HSF1 and HSF2 dissociated from the granules and both proteins were
partly translocated into the cytosol (Fig.
7A). After a second heat shock of 30 minutes or 1 hour, a decrease
in granule-positive cells was detected, because almost 100% of primarily
heat-shocked cells, but less than 15% of thermotolerant cells, exposed to a
1-hour heat shock contained HSF-positive granules
(Fig. 7B). Because HSF1 and
HSF2 behaved identically, only HSF1 was used as a marker for nuclear stress
granules in the following experiments.
|
Nucleolar localization of Hsp70 coincides with the localization of
HSF1 in stress granules
One of the mechanisms regulating thermotolerance and attenuation of the
heat shock response is the inhibition of HSF1 activity by molecular chaperones
such as Hsp70 and Hsp90 and their co-chaperones
(Morimoto, 2002). However, the
role of these chaperones in the regulation of nuclear stress granules has not
been elucidated. Using immunoprecipitation of Myc-tagged HSF1 from stably
transfected 4H9 cells after methionine labeling followed by identification of
the interacting proteins by mass spectrometry, we found Hsc70 and Hsp70
forming a complex with HSF1 in a stoichiometric manner
(Fig. 8A). During attenuation
of the heat-shock response and in thermotolerant HeLa cells, dynamically
regulated complex formation was detected between HSF1 and Hsp70
(Fig. 8B). At the control
temperature and upon heat shock of less than 30 minutes, HSF1 was found in a
complex with Hsp70, whereas this complex disassembled after 30 minutes and was
hardly detectable at 2 hours (Fig.
8B, data not shown). After 2 hours of continuous heat shock, the
interaction began to increase. The interaction pattern with Hsp70 correlated
well with the onset of attenuation seen previously in HSF1 DNA-binding
activity and phosphorylation, as well as with the decline in granule-positive
cells (Fig. 1). During recovery
periods, complex formation between HSF1 and Hsp70 gradually increased
(Fig. 8B).
Next, we investigated how the subcellular localization of Hsp70
corresponded to the interaction pattern seen in
Fig. 8B and to the heat-shock
granule formation. Hsp70 and its cochaperones such as Hsp40 are rapidly
translocated from the cytosol to the nucleoli upon heat shock
(Welch and Feramisco, 1984;
Welch and Suhan, 1986
; Hattori
et al., 1993) but the significance of this rapid change in subcellular
distribution is poorly understood. The localization of HSF1 and Hsp70 was
investigated by double-staining immunofluorescence microscopy and, at normal
temperature, HSF1 was found both in the cytosol and nucleus, whereas Hsp70 was
mainly cytosolic (Fig. 8C).
Upon heat shock of less than 1 hour, the increasing intensity and size of the
stress granules coincided with the ongoing translocation of Hsp70 into the
nucleoli (data not shown). At 1 hour, HSF1 was primarily detected in mature
nuclear granules and Hsp70 concentrated in the nucleoli
(Fig. 8C). Interestingly, at
this time point, the interaction between HSF1 and Hsp70 was dramatically
downregulated (Fig. 8A). During
the attenuation of the heat-shock response (2-6 hours of continuous heat
shock), both Hsp70 and HSF1 gradually dissociated from nucleoli and the stress
granules, respectively, and HSF1-Hsp70 complex formation increased
(Fig. 8A,C, data not shown). In
thermotolerant cells, HSF1 and Hsp70 formed an interacting complex and were
both mainly localized in the cytosol (HS+3R;
Fig. 8A,C). Upon exposure of
thermotolerant cells to a second heat shock, a high level of interaction
persisted and HSF1-positive granules were detected only in cells in which
Hsp70 was translocated into nucleoli (HS+3R+HS1h;
Fig. 8C). The number of cells
containing HSF1 in stress granules and Hsp70-positive nucleoli corresponded to
the heat-shocked thermotolerant cells shown in
Fig. 7B (data not shown). In
these experiments, the nucleolar localization of Hsp70 coincided with the
downregulation of HSF1-Hsp70 interaction and localization of HSF1 in nuclear
stress granules. The reversible localization pattern of Hsp70 into and out of
the nucleoli appeared to be closely related to the subcellular distribution of
HSF1, and could thereby contribute to the regulation of HSF1 activity.
![]() |
Discussion |
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In addition to the co-localization of HSF1 and HSF2 in nuclear stress
granules, we provide the first in vivo evidence of complex formation between
HSF1 and HSF2. Our data reveal a regulated interaction between HSF1 and HSF2
isoforms during the course of heat shock, possibly caused by regulated
solubility of different HSF2 isoforms, as indicated by Mathew et al.
(Mathew et al., 2001). We also
show that the HSF1-HSF2 complex is efficiently disassembled in thermotolerant
cells, suggesting modifications in the properties of the HSF1-interacting
protein complex. Further experiments are needed to reveal whether the
downregulation of HSF1-HSF2 interaction is involved in the suppression of HSF1
activity during thermotolerance, and how the HSF2 isoform composition reflects
the overall heat shock response. By analyzing a set of HSF2 deletion mutants,
we show that the physical interaction with HSF1 requires an intact HR-A/B
oligomerization domain of HSF2. It is still an open question whether HSF1 and
HSF2 form a heterodimer or heterotrimer, or whether the interaction occurs
between two homo-oligomers. Considering our results and the structural
similarity, it is likely that HSF1 and HSF2 form a hetero-oligomer.
Despite the prominent interaction between HSF1 and HSF2, we failed to
detect HSF2 in the heat-shock-induced HSE-binding complex, which is in
agreement with Mathew et al. (Mathew et
al., 2001). Because these observations indicate that heat stress
does not activate HSF2 HSE-binding activity, the HSF1-HSF2 complex formation
together with the subnuclear co-localization represents a previously
uncharacterized step in the regulation of the stress-signaling pathway in
human cells. To address the question of whether HSF1-HSF2 interaction affects
the localization of HSF1 in stress granules, we performed double-staining
experiments using the HSF2 deletion mutants. Strikingly, expression of HSF2
mutant lacking the DNA-binding domain (HSF2 108-517) inhibited the recruitment
of HSF1 into the stress granules, which could be due to the physical
interaction between this mutant and HSF1. An attractive explanation is that
the effect caused by HSF2 108-517 would lead to a defect in the interaction
between the chromosomal nucleation sites and the HSF1-HSF2 108-517
heterocomplex. This is in good agreement with another study, in which the
granule-forming-ability of HSF1 was shown to be strictly dependent on the
DNA-binding and the oligomerization domains of HSF1
(Jolly et al., 2002
). By
contrast, neither of the deletion mutants lacking the HR-A/B oligomerization
domain (HSF2 203-517, HSF2
126-201) was able to form a complex with
HSF1 and thereby affect the compartmentalization of HSF1 in the stress
granules. Therefore, the capability of HSF2 to oligomerize seems to be crucial
for both interaction with HSF1 and localization in the stress granules.
Unexpectedly, the C-terminal deletion mutant (HSF2 1-396), which both
interacted and co-localized with HSF1, was spontaneously translocated into the
stress granules with endogenous HSF1. This further emphasizes the
interdependency between HSF1 and HSF2 in their stress granule
localization.
A characteristic feature of the heat-shock response is acquired upon
recovery from stressful conditions. The mechanisms regulating thermotolerance
have been closely related to the inhibition of HSF1 by direct interaction
between upregulated Hsp70 and its co-chaperones
(Morimoto, 2002). Owing to the
changes in HSF1 regulation upon acquired thermotolerance, we wanted to examine
the localization of HSF1 and HSF2 in nuclear stress granules under these
conditions. Our results demonstrate a suppression of HSF1 and HSF2
localization in stress granules in heat-shocked thermotolerant cells, which
correlates well with the inhibition of HSF1 activity during thermotolerance
and suggests a role for Hsps in the regulation of granule formation. We also
show that the translocation of Hsp70 into and out of the nucleoli coincides
with the localization of HSF1 to nuclear stress granules and interaction with
HSF1. The dissociation of the HSF1-Hsp70 complex during heat shock corresponds
to the translocation of Hsp70 into nucleoli. Simultaneously, the DNA-binding
activity and hyperphosphorylation of HSF1, and the number of granule-positive
cells are at maximum. During the attenuation of HSF1 activity, Hsp70
dissociates from the nucleoli, the complex formation between Hsp70 and HSF1
increases, and HSF1 relocates from the stress granules. Concomitant with the
inhibition of HSF1 localization to granules in thermotolerant cells exposed to
a second heat shock, fewer cells are observed that contain Hsp70 in their
nucleoli. Taken together, our results show a strict correlation between the
subnuclear compartmentalization of HSF1 and Hsp70 nucleolar localization, and,
because the dissociation of HSF1 from stress granules during attenuation also
coincides the translocation of Hsp70 out from the nucleoli, it is plausible
that the nucleolar localization of Hsp70 contributes to the regulation of
HSF1.
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Acknowledgments |
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Footnotes |
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Present address: Department of Cell Biology, University of Oklahoma Health
Sciences Center, Oklahoma City, OK-73104, USA.
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References |
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