From the Department of Adult Oncology, Dana Farber
Cancer Institute and Joint Center for Radiation Therapy, Harvard
Medical School, Boston, Massachusetts 02115 and the
CNRS
EP 560, Institut Pasteur de Lille, 1 Rue
Calmette-BP 245, 59021 Lille Cedex, France
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ABSTRACT |
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Heat shock factor 1 (HSF1) is the key
transcriptional regulator of the heat shock genes that protect cells
from environmental stress. However, because heat shock gene expression
is deleterious to growth and development, we have examined mechanisms
for HSF1 repression at growth temperatures, focusing on the role of
phosphorylation. Mitogen-activated protein kinases (MAPKs) of the ERK
family phosphorylate HSF1 and represses transcriptional function. The
mechanism of repression involves initial phosphorylation by MAP kinase
on serine 307, which primes HSF1 for secondary phosphorylation by
glycogen synthase kinase 3 on a key residue in repression (serine 303). In vivo expression of glycogen synthase kinase 3 ( or
) thus represses HSF1 through phosphorylation of serine 303. HSF1 is also phosphorylated by MAPK in vitro on a second residue
(serine 363) adjacent to activation domain 1, and this residue is
additionally phosphorylated by protein kinase C. In vivo,
HSF1 is repressed through phosphorylation of this residue by protein
kinase C
or -
but not MAPK. Regulation at 37 °C, therefore,
involves the action of three protein kinase cascades that repress HSF1
through phosphorylation of serine residues 303, 307, and 363 and may
promote growth by suppressing the heat shock response.
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INTRODUCTION |
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Exposure of cells to elevated temperatures leads to the expression of the heat shock response, in which the induction of a cohort of heat shock proteins (HSPs)1 is accompanied by the expression of heat resistance (1, 2). In mammalian cells, HSP genes are regulated at the transcriptional level by heat shock factor 1 (HSF1), a sequence-specific transcription factor that interacts with heat shock elements (HSEs) in their promoters (3-5). Much evidence now suggests that although HSPs protect cells during hyperthermia, growth of cells under nonstress conditions is incompatible with the expression of the heat shock response (6). Heat shock arrests cells in G1 (7, 8), and HSF1 expression delays the progression of cells through G1,2 whereas HSP70 overexpression inhibits growth and development (6, 9). In addition, HSF1 represses the ability of Ras protein to activate the promoters of immediate early genes such as c-fos (10). There evidently exists, however, a well-conserved mechanism to inhibit the activity of HSF1 at 37 °C and prevent these deleterious effects on cell growth. We have examined molecular mechanisms involved in the repression of HSF1 by signaling pathways involved in growth regulation.
Activation of HSF1 involves the conversion of HSF1 from a latent
cytoplasmic monomer to a trimeric nuclear protein complex that controls
the transcription of heat shock genes (3-5, 11) Trimerization is
governed by arrays of amphipathic helical residues ("leucine
zippers") in the N-terminal domain and is negatively regulated by a
fourth such domain in the C terminus (3, 12). However, although
necessary, nuclear localization and DNA binding are not sufficient for
the full transcriptional competence of HSF1, which can be activated to
an intermediate state in which it binds to HSE sequences but does not
stimulate transcription (13-16). Much evidence suggests a role for
phosphorylation in the conversion of HSF1 from this intermediate state
into a transcriptionally active form (13, 14, 16, 17). In addition, HSF
from yeast and HSF1 from mammalian cells both undergo
hyperphosphorylation during heat shock, and their hyperphosphorylation
correlates well with transcriptional activation (17-19). Our previous
studies showed that HSF1 is phosphorylated at multiple sites mostly on
serine residues and that mitogen-activated protein kinases (MAPKs) of the ERK-1 family phosphorylate HSF1 on serine and repress the transcriptional activation of the heat shock protein 70B
(HSP70B) promoter by HSF1 in vivo (20). These
experiments and a number of other reports thus indicate that HSF1 is
antagonized by Ras-MAPK signaling and that this may be a mechanism for
HSF1 repression at 37 °C (20-24). The repressive effects of MAPK
were transmitted through a specific serine residue (Ser-303) in a
proline-rich sequence within the transcriptional regulatory domain of
human HSF1 (20). However, despite the importance of Ser-303 in
transmitting the signal from the MAPK cascade to HSP70 transcription,
there was no evidence that Ser-303 could be phosphorylated by MAPK
in vitro, although an adjacent residue (Ser-307) was avidly
phosphorylated by MAPK (20). Preliminary studies suggested that Ser-303
is phosphorylated by glycogen synthase kinase 3 (GSK3) through a mechanism dependent on primary phosphorylation of Ser-307 by MAPK (20).
In the present experiments, we have demonstrated the sequential phosphorylation by MAPK and GSK3 and its role in HSF1 repression.
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MATERIALS AND METHODS |
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Cell Culture-- NIH 3T3 cells were grown to confluence in Dulbecco's modified Eagle's medium containing 10% bovine calf serum and passaged at a 1/10 ratio.
Site-directed Mutagenesis, Expression, and Purification of
Recombinant HSF1--
Oligonucleotide-directed mutagenesis was
performed using the pALTER-1 vector as described previously (20).
Following mutation, DNA sequences were checked by dideoxynucleotide
sequencing and cloned into the pcDNA3.1 vector for mammalian
expression and into the pET22B vector for expression in
Escherichia coli. For purification, wild-type human HSF1
cDNA and point-mutated forms S303G, S307G, and S363G (25) were
induced in E. coli by
isopropyl-1-thio--D-galactopyranoside treatment,
extracted, and purified to homogeneity as described (26).
In Vitro Phosphorylation of Recombinant HSF1--
Purified MAPK
from P. ochraceus (p44mpk), PKC
from rabbit brain and GSK3
, mitogen-activated protein
kinase-activated protein kinase 2 (MAPKAP K2), and pp90rsk
S6 kinase isoform RSK2 from rabbit muscle were obtained from Upstate
Biotechnology (Lake Placid, NY). In vitro phosphorylation of
HSF1 by MAP kinase and GSK3 was carried out as described previously (20). Purified enzymes (MAPK and GSK3
) were tested for contamination with other kinases that might phosphorylate HSF1 by assaying with peptides specific for MAPKAP K2, RSK2, and PKC
. RSK2 activity was
assayed as described using substrate peptide RRRLSSRA, utilizing purified RSK2 enzyme as a positive control (27). Assays were carried
out with or without MAPK addition, because MAPK is a potent activator
of RSK2. MAPKAP K2 activity in the GSK3
preparation (with or without
the addition of MAPK) was assayed as for RSK2 using MAPKAP K2 substrate
peptide (KKLNRTLSVA) (28). Peptides were purified to greater than 90%
by high performance liquid chromatography. Neither the MAPK nor GSK3
preparation was significantly contaminated with MAPKAP K2, RSK2, or
PKC
. HSF1 can also be phosphorylated in vitro with casein
kinase 2 and calmodulin kinase II; however, the phosphopeptide maps
obtained from HSF1 treated with either enzyme were completely different
from the MAPK or GSK3
maps (data not shown), indicating that HSF1
phosphorylation with MAPK or GSK3
does not reflect contamination
with either casein kinase 2 or calmodulin kinase II. To phosphorylate
HSF1 with PKC
, 5 µg of HSF1 was incubated with enzyme (25 ng),
phosphatidylserine (100 µg/ml), CaCl2 (1 mM),
ATP (5 µM), and 32P-labeled ATP (1 µCi/µl) in buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, and 0.2 mM EGTA) to a
final volume of 50 µl. Incubations were for 12 min at 25 °C.
Protein kinase C activity was assayed using the peptide
QKRPSQRSKYL.
Two-dimensional-Phosphopeptide Mapping and Peptide Sequencing-- Two-dimensional phosphopeptide mapping was carried out after digestion with trypsin as described previously (20, 28). For sequencing, peptides were eluted from the TLC medium and subjected to multiple cycles of Edman degradation, and amino acids were identified by high performance liquid chromatography elution time (29). Sequencing was performed by the Biopolymer Laboratory, Brigham & Women's Hospital, Boston, MA.
Electrophoretic Mobility Shift Assay for HSF1-HSE Binding-- Binding of rHSF1 or nuclear HSF1 to HSE was assayed by electrophoretic mobility shift assay as described previously (14, 30).
Transfection--
HSF1 constructs were co-transfected with the
chloramphenicol acetyltransferase (CAT) construct p2500CAT, which
contains 2.5 kilobase pairs of 5'-noncoding sequence from the
heat-inducible human HSP70B gene (31) (StressGen, Victoria,
British Columbia, Canada). Cells were seeded at a density of 250,000 per 100-mm tissue culture dish 24 h prior to transfection carried
out using calcium phosphate precipitation according to the
manufacturer's protocol (Promega). Cells were harvested 48 h
after incubation with plasmids for assay of CAT protein expression
(20). To control for transfection efficiency, cells were co-transfected
with pCMV--galactosidase plasmid and assayed for
-galactosidase
as described (10). In some experiments, HSF1 was co-transfected with
MEK-1 expression vector (pCMV-MKK-1) from Dr. N. G. Ahn
(University of Colorado, Boulder, CO) (32). For GSK3 plasmids
pMT2GSK3
and pMT2GSK3
(33), we thank Dr. J. R. Woodgett
(Ontario Cancer Institute), and for pPKC
and pPKC
expression
plasmids and purified recombinant hGSK3
, we thank Dr. B. Price, Dana
Farber Cancer Institute (Boston, MA).
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RESULTS |
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In order to investigate the hypothesis that HSF1 is phosphorylated
on the key regulatory serine residue 303 by GSK3 only after a priming
phosphorylation by MAPK, we first examined the effect of mutating
serines 303 and 307 on sequential phosphorylation by MAPK and GSK3
(Fig. 1). Wild-type recombinant HSF1
(wtHSF1) was not phosphorylated by recombinant GSK3 (Fig 1,
lane 3) as shown previously with purified rabbit GSK3
(20) but was phosphorylated by purified GSK3
(lane 2).
MAPK phosphorylated wtHSF1 (Fig. 1, lane 1) and led to a
marked increase in phosphorylation by GSK3
and -
(lanes
4 and 5). When similar experiments were carried out on
HSF1 with a serine to glycine mutation (S303G), the levels of
phosphorylation in incubations with MAPK, GSK3
, and GSK3
were
similar to those in the wt control when used individually. (Fig. 1,
lanes 6-8). However, we did not observe increased
phosphorylation by GSK3
and -
when combined with MAPK
(lanes 9 and 10). Thus, inactivation of the
putative GSK3 site (Ser-303) prevents MAPK stimulation of HSF1
phosphorylation by GSK3 (Fig. 1). The phosphorylation of HSF1 observed
with GSK3
alone, without MAPK priming, is evidently at residues
other than Ser-303 as similar levels of 32P uptake were
observed in wtHSF1 and S303G when incubated with GSK3
(Fig. 1,
lanes 2 and 7). Substitution of glycine for
serine at Ser-307 (S307G) led to a HSF1 with similar properties to
S303G, with phosphorylation by MAPK and GSK3
(Fig. 1, lanes
11 and 12), but no stimulation of GSK3 induced
phosphorylation by MAPK (lanes 14 and 15); this
indicates a requirement for intact Ser-307 and MAPK for GSK3
phosphorylation of HSF1 at Ser-303, consistent with a priming role for
MAPK in HSF1 regulation by GSK3 (20). The experiments also show that
despite loss of the phosphorylation site at serine 307, S307G is still
phosphorylated by MAPK (lane 11). This is, however,
consistent with previous studies indicating two major MAPK sites
on HSF1 (Fig. 1; Ref. 20).
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In order to confirm that the changes in levels of 32P
incorporation observed in HSF1 treated sequentially with MAPK and
GSK3 (Fig. 1) were due to hierarchical phosphorylation, we examined the relative levels of phosphorylation of Ser-303 and Ser-307 using
tryptic two-dimensional phosphopeptide mapping (Fig. 2). We used
GSK3
in this study due to its more ready availability. Incubation of
HSF1 with purified MAPK in vitro followed by two-dimensional mapping led to the resolution of two major phosphopeptides (Fig. 2A, a and b) as shown previously (20). A number
of minor spots were also seen. Two of these, which occur at positions
to the left of spots a and b in Fig. 2, were
observed in most experiments and may correspond to higher
phosphorylated forms of peptides a and b, with similar hydrophobicity
to a and b but with lower electrophoretic mobility due to their greater
negative charge (28). The spots closer to the origin were not observed
consistently and may be products of incomplete digestion or minor
phosphorylation (Fig. 1A). Although incubation in
vitro with GSK3
did lead to phosphorylation of HSF1 on residues
other than Ser-303 (Fig. 1), no clear spots were resolved by
two-dimensional mapping (not shown). Treatment of HSF1 with GSK3
after preincubation with MAPK led to increased phosphorylation (Fig. 1)
and caused the appearance of a new spot (spot c) and the concomitant
depletion of spot a (Fig. 2B). These experiments therefore
suggest that spot c is produced through the phosphorylation by GSK3
of the polypeptide sequence in HSF1 that migrates as spot a and that
the resulting spot (c) is retarded in the electrophoretic field
(relative to a) due to the increase in negative charge resulting from
double phosphorylation of this sequence (Fig. 2B). A
previous study using purified rabbit GSK3
had indicated the
phosphorylation of HSF1 on four sites in addition to peptide c
(phosphopeptides w, x, y, and z) by GSK3
(20). This appeared to be
due to contamination of the GSK3
used in that study with another
kinase (MAPKAP K2), which is activated by MAP kinase. We have
subsequently shown that highly purified MAPKAP K2 phosphorylates HSF1
on sites w-z and that purified rabbit GSK3
and human recombinant
GSK3
, which are both free of MAPKAP K2 contamination, do not (data
not shown). Our previous studies showed that the peptide that migrates
to form spot a comprises amino acids 299-309 (EEPPSPPQSPR), containing sites at position 307 for MAPK phosphotransferase activity and a
potential site at position 303 for GSK3 (20). Spot a thus consists of
amino acids 299-309 singly phosphorylated at Ser-307, and it is
predicted that spot c is doubly phosphorylated, at Ser-307 by MAPK and
at Ser-303 by GSK3
. Mutation of Ser-307 to glycine (S307G) abolished
both spot a and spot c after serial treatment with MAPK and GSK3
(Fig. 2C). Mutation of HSF1 at Ser-303 (S303G) abolished the
appearance of spot c and prevented the depletion of spot a after serial
phosphorylation, indicating that spot c is the product of double
phosphorylation by MAPK and GSK3
and that Ser-303 is phosphorylated
by GSK3
(Fig. 2D). These experiments are consistent with
the hypothesis of primary phosphorylation of HSF1 on Ser-307 being a
prerequisite for secondary phosphorylation on Ser by GSK3
.
We next examined the potential role of GSK3 in the regulation of HSF1
activity in vivo. We used a system described previously in
which HSF1 expression from the pcDNA3.1.HSF1 (pHSF1) vector activates a heat shock promoter reporter construct in the absence of
heat shock (20, 30, 31). HSF1 expression strongly induced HSP70B promoter activity in NIH3T3 cells, whereas
co-expression of a GSK3 expression vector repressed the effects of
HSF1 by 80%, and co-expression of pMEK-1, which increases cellular
MEK-1 levels and specifically activates ERK1 and ERK2 (20), further reduced activity (Fig. 3). Similar
results were obtained with a GSK3
expression vector (not shown).
When similar experiments were carried out using HSF1 mutant S303G, we
found that repression of HSP70B promoter activity by GSK3
was effectively abolished, identifying serine 303 as the target for
HSF1 repression by GSK3
in vivo (Fig. 3). Neither
mutation of HSF1 at Ser-303 or Ser-307 nor treatment with GSK3
or
MAPK affected the binding of HSF1 to heat shock elements, indicating
that the effects observed are exerted downstream of DNA binding, at the
level of transcriptional transactivation (20) (see Fig. 7). However,
Ser-303 mutation did not completely block the repression of HSF1 by
MEK1, suggesting further effects of MAPK on HSF1 function (Fig. 3).
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Because the data in Fig. 3 suggest that MAPK exerts repressive effects
on HSF1 in addition to those mediated through Ser-303, we next analyzed
the second major site on HSF1 phosphorylated by MAPK in
vitro. We showed in Fig. 1A that two major
phosphopeptides, a and b, are resolved in HSF1 after MAPK treatment
in vitro. Phosphopeptide a contains serines 303 and 307 (Fig. 1). Analysis of phosphopeptide b isolated from the TLC plates
after two-dimensional mapping indicated a sequence correspond to amino
acids 362-372 in the human HSF1 sequence (25). To confirm this
finding, we carried out site-directed mutagenesis on serine 363 contained within phosphopeptide b, which forms part of a sequence
(PPSP) that conforms to a MAPK consensus motif (PX(S*/T*)P (where the
asterisk indicates the phosphorylation of a threonine or serine residue
(34-36)). Mutation of Ser-363 to alanine, to create S363A, had a
profound effect on the phosphopeptide map of HSF1 after MAPK
phosphorylation and major spot b was eliminated (although a faint spot
was detected in some experiments) (Fig. 4). The peptide sequence contains other
serine/threonine residues (notably, amino acids 368 and 369) within a
proline-rich region that could potentially be phosphorylated at a low
rate by MAPK. These experiments implicate Ser-363 as the second residue
in HSF1 that undergoes phosphorylation after treatment with MAPK
in vitro. To investigate the functional importance of
Ser-363, we next examined the ability of MEK-1 expression to repress
transcriptional activation of the HSP70B promoter by the
S363A mutant (Fig. 5). Contrary to
expectations, although Ser-363 is avidly phosphorylated by MAPK
in vitro (Figs. 1 and 4), the S363A mutant was equally
sensitive to MEK-1 repression compared with the wt control (Fig. 5).
Thus, either Ser-363 phosphorylation does not affect the
transcriptional activity of HSF1 or this site is not phosphorylated by
MAPK in vivo. This region in HSF1 (360-365; RPPSPP)
contains overlapping consensus motifs for both MAPK (PXS*/T*P) and PKC
(RXXS*/T*) (36). We therefore examined the effect of expressing two
isoforms of protein kinase C (PKC and PKC
) on the activities of
co-transfected wtHSF1 and S363A (37). Recent studies have shown the
existence of at least 11 PKC family members, which belong to three
distinct classes based on structure and responsiveness to activators:
PKC isoforms
,
1,
II, and
are Ca2+- and
phorbol ester-dependent; PKC isoforms
,
,
, and
require phorbol esters but are Ca2+-independent; and
the atypical isoforms
and
require neither activator (38, 39).
We chose PKC
and PKC
as representative members of the family,
ranging from the "classical" Ca2+- and phorbol
ester-dependent PKC
to the atypical PKC
, which is
dependent on neither factor (37, 38). The transcriptional activity of
wtHSF1 was inhibited by overexpression of MEK1, PKC
, or PKC
(Fig.
5). One explanation for this finding could be that PKC acts indirectly
through upstream activation of MAPK family members ERK1 and ERK2 (40).
However, on examining the effects of S363A mutation on responses to PKC
expression, we found that although it is not involved in inhibition by
MEK1, S363A substitution prevented repression of HSF1 activity caused
by high level expression of PKC
or PKC
(Fig. 5). Mutation of HSF1
in the 303 position (S303G) did not, however, prevent HSF1 repression
in cells overexpressing PKC
, indicating that the effects of PKC are
exerted either directly or indirectly through phosphorylation of
Ser-363 and do not involve Ser-303 (Fig.
6). To further probe the potential
effects of PKC on HSF1 activity, we used calphostin C, which binds to
the regulatory domain of PKC isoforms and specifically inhibits their
activity (41). Incubation of cells co-transfected with pHSF1 and
pPKC
with calphostin C partially restored HSF1 activity, further
implying a role for PKC
in HSF1 repression (Fig. 6). However,
incubation with calphostin C did not markedly enhance HSF1 activity in
the absence of PKC
co-transfection in these cells (Fig. 6). Thus, Ser-363, although avidly phosphorylated by purified MAPK in
vitro, is not a major target for MAPK in vivo but
instead mediates HSF1 regulation by members of the PKC family (Figs. 5
and 6). We next determined whether HSF1 is directly phosphorylated by
PKC
(Fig. 7). Recombinant HSF1 was
phosphorylated by PKC
in vitro, whereas the
phosphorylation of purified S363A under identical conditions was
markedly reduced, suggesting that Ser-363 is phosphorylated by PKC
and directly mediates the effects of PKC expression on the
transcriptional activity of HSF1 (Figs. 6 and 7).
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Finally, in order to examine the effects of GSK3 and PKC
expression on the ability of HSF1 to form nuclear trimers capable of
binding to heat shock elements, we carried out electrophoretic mobility
shift assay analysis on nuclear extracts from control cells and
transfectants (Fig. 8). HSF1 expression
(Fig. 8, lane 5) led to the formation of a complex of
similar electrophoretic mobility to HSF1-HSE complexes from heat
shocked cells (Fig. 8, lanes 2 and 3). It is
notable that although HSF1 expression increases activity of the
HSP70B promoter by at least 100-fold, activation of HSF1-HSE
binding increases only 2-3-fold in the transfectants (Figs. 3 and 7).
This reflects the efficiency of transfection in the system used here
(0.5-1.0%). Thus, increases in HSF1 binding in the transfectants are
diluted by the 100-fold excess of untransfected cells. The
trans-activation assay reports only on cells that have been
co-transfected with HSF1 expression plasmid and the HSP70B promoter reporter construct and is unaffected by the presence of
untransfected cells. Co-expression of GSK3
and PKC
did not inhibit the formation of the HSF1-HSE complex (Fig. 8, lanes
6 and 7). Thus, overexpression of these protein kinases
represses HSF1 activity at step other than formation of DNA binding
nuclear trimers.
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DISCUSSION |
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These experiments demonstrate that HSF1 is phosphorylated by
GSK3 on a residue (Ser-303) within the transcriptional regulatory domain (Fig. 1) (42, 43). In addition, HSF1 phosphorylation at Ser-303
by GSK3
was dependent on HSF1 phosphorylation by MAPK on an adjacent
residue (Ser-307) (Figs. 1 and 2). HSF1 phosphorylation at Ser-303 thus
involves hierarchical phosphorylation with primary phosphorylation by
MAPK preceding secondary modification by GSK3. A similar indirect
mechanism was demonstrated previously in the phosphorylation of protein
substrates by GSK3 (44-46). Consistent with previous experiments
showing that transfection of activated Ras protein and MEK-1 represses
the heat shock response and that dominant negative constructs of ERK1
are activating, our data suggest that HSF1 is repressed under growth
conditions through the Ras-MAPK pathway by primary phosphorylation by
MAPK, leading to secondary phosphorylation by GSK3 (20-24). In
addition, the finding that overexpression of GSK3
or GSK3
directly represses HSF1 suggests that a subpopulation of HSF1 molecules
may be constitutively phosphorylated at Ser-307 and that HSF1 may be
directly regulated by the GSK3 pathway as well as indirectly through
the Ras-MAPK pathway (Figs. 2 and 3). GSK3
or GSK3
is involved in
transcriptional regulation and is regulated by a signaling pathway
activated by cell surface receptors for the Wnt proteins and propagated
through a kinase cascade involving phosphatidylinositol 3-kinase and
protein kinase B (46-49). This process results in the inhibition of
GSK3 activity, which is constitutive in noninduced conditions (48). Protein kinase B (also known as RAC-PK or Akt) is activated by heat
shock and could potentially activate HSF1 through GSK3 inhibition (50).
It is not clear whether one or both of the isoforms of GSK3 interact
with HSF1 in vivo because both GSK3
and GSK3
can phosphorylate HSF1 in vitro and repress HSF1 when expressed
at high level in vivo (Figs. 1 and 2) (20, 33). It is also
apparent that HSF1 repression by MEK-1 involves mechanisms in addition to priming HSF1 for GSK3 phosphorylation (Fig. 2). We therefore examined the role in HSF1 repression of a second residue phosphorylated by MAPK in vitro located at serine 363 within a MAPK
consensus motif (Figs. 2 and 4). The analysis indicated, however, that
Ser-363 is not involved in repression of HSF1 by the Ras-MAPK pathway, and in fact, Ser-363 mediates HSF1 repression by the PKC family (Figs.
5 and 6). PKC
and PKC
both strongly repressed HSF1 function, and
repression was relieved by mutation of Ser-363 but not by mutation of
Ser-303 (Figs. 5 and 6). The findings that PKC phosphorylates HSF1
in vitro, that this effect is inhibited by loss of Ser-363, and that the repressive effects of PKC
expression are reversed by
either S363A mutation or calphostin C exposure suggest that PKC
represses HSF1 largely through direct enzymatic modification (Figs.
5-7). Previous studies suggested a potential role for PKC in the heat
shock response based on findings that phorbol esters enhance HSF1-HSE
binding and HSP synthesis during heat shock (51). Similar observations
were made in our unpublished studies and are seemingly at odds with a
role for PKC in HSF1
repression.3 However, in the
NIH 3T3 cell line used here, exposure to the active phorbol ester
phorbol myristate acetate (PMA) leads to a progressive down-regulation
of PMA binding activity, reaching levels only 20% of controls by
8 h exposure to 10
7 M
PMA.4 In addition, such
treatment leads to functional loss of PKC activity, as indicated by the
finding that long term treatment with PMA eliminates subsequent
PMA-induced MAPK induction in these cells (40). Stimulatory effects of
PMA on HSF1 activity may thus reflect the down-regulation of
PMA-binding PKC species. In addition, PMA has been shown to increase
the cellular levels of HSF1 and HSF1 mRNA (52). Effects of PMA on
HSF1 activity may therefore also reflect alterations in HSF1 levels.
However, our findings indicate that directly activating PKC by
overexpression leads to HSF1 repression through phosphorylation on
Ser-363 (Fig. 6). It is not clear, however, to what extent PKC is
involved in HSF1 regulation under basal conditions. Treatment with
calphostin C caused only a slight increase in HSF1 activity in NIH 3T3
cells (Fig. 6), although a larger (50%) increase was observed in HeLa
cells (not shown). PKC may thus play a less prominent role, compared
with MAPK in HSF1 repression during conditions of continuous growth
(20). Repression through Ser-363 may be more significant in conditions leading to acute increases in PKC activity, such as the activation of
serpentine receptors and binding of stimulatory ligands to growth
factor receptors (38). It is apparent however, that the region in HSF1
containing Ser-363 is phosphorylated in cells in vivo,
suggesting a potential role for Ser-363 in HSF1 regulation at 37 °C
(20).
Repression by MAPK and GSK3 involves HSF1 phosphorylation at sites within the regulatory domain that control the activity of adjacent activation domains (42, 43). PKC repression is, however, exerted through a site (Ser-363) in a previously uncharacterized region of HSF1 between the regulatory domain (amino acids 220-310) and the C-terminal activation domains (amino acids 371-529) (42, 43). The close proximity of Ser-363 to activation domain 1 (amino acids 371-430) suggests a potential regulatory interaction (42, 43). Although we have not addressed here the role of phosphorylation in HSF1 activation by heat shock, these experiments suggest possible mechanisms. HSF1 activation by heat shock could involve antagonism of the mechanisms that repress HSF1 at 37 °C, an overriding regulatory change imposed by heat shock, or a combination of both mechanisms. That the reversal of negative regulation during heat shock is a potential mechanism for HSF1 activation is suggested by our findings that HSF1 overexpression in the absence of heat shock activates its function (Fig. 2) (10, 20, 30). These findings imply that the elevated HSF1 concentrations in transfectants titrate intracellular repressors and thus permit HSF1 activation at high concentrations. In addition, HSF1 mutants resistant to the inhibitory phosphorylations at Ser-303 and Ser-307 activate transcription at lower concentrations than those required for HSP70 promoter activation by wild-type HSF1, again suggesting the existence of titratable HSF1 inhibitors (20). However, our earlier studies suggested that HSF1 is phosphorylated in vivo on sites associated with HSF1 repression (Ser-303/307; peptide a) and Ser-363 (peptide b) before and after a heat shock (30 min at 42 °C) that activates HSP gene transcription (20). Thus, dephosphorylation of these residues may not be essential for HSF1 activation by heat shock. Therefore, although reversal of HSF1 repression may participate in activation, it seems likely that additional events unique to heat shock are involved in the full activation of HSF1 by heat, as discussed previously (17). A similar conclusion was reached in previous studies of the transcriptional regulatory domain of HSF1 showing that this domain has the property of dominantly repressing transcriptional activation domains at 37 °C and activating such domains during heat shock (43). The degree of transcriptional activation induced by heat shock exceeded the amount predicted to be caused by the reversal of repression (43).
In summary, therefore, HSF1 is a tightly regulated factor repressed at 37 °C by the action of protein kinase cascades terminating in the activation of MAPK, GSK3, and PKC, which lead to phosphorylation of inhibitory serine residues 307, 303, and 363. Coupling HSF1 repression to protein kinase activities associated with normal anabolic function may ensure suppression of HSF1 at 37 °C during growth and recovery from stress.
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ACKNOWLEDGEMENTS |
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We thank Dr. C. N. Coleman and the Associates of the Joint Center for Radiation Therapy for encouragement and support. We are grateful to Margaret Condron and David Teplow for peptide sequencing, to David Sachs for help and advice related to two-dimensional mapping, and to James Woodgett for discussions regarding GSK3. For advice regarding the transfection experiments, we thank Yue Xie and Changmin Chen.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA4707, CA31303, and CA50642.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Biology, University of Massachusetts, Lowell, MA 01854.
¶ These authors contributed equally to this work.
** To whom correspondence should be addressed: Dept. of Adult Oncology, Dana Farber Cancer Institute and Joint Center for Radiation Therapy, Harvard Medical School, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3885; Fax: 617-632-4599; E-mail: stuart_calderwood{at}dfci.harvard.edu.
1 The abbreviations used are: HSP, heat shock protein; HSF, heat shock factor; HSE, heat shock element; GSK, glycogen synthase kinase; MAPK, mitogen-activated protein kinase; MAPKAP K2, MAPK-activated protein kinase 2; CAT, chloramphenicol acetyltransferase; PMA, phorbol myristate acetate; wt, wild-type; PKC, protein kinase C.
2 J. L. Bruce, C. Chen, Y. Xie, R. Zhong, M. A. Stevenson, and S. K. Calderwood, submitted for publication.
3 X. Zhang and S. K. Calderwood, unpublished data.
4 B. Farnum and S. K. Calderwood, unpublished data.
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