From the Department of Molecular and Cellular
Biochemistry and
Department of Physiology, Chandler
Medical Center, University of Kentucky,
Lexington, Kentucky 405036-0298, and the ¶ Department of
Biochemistry and Molecular Biology, School of Hygiene and Public
Health, Johns Hopkins University, Baltimore, Maryland 21205
Received for publication, September 4, 2000, and in revised form, February 1, 2001
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ABSTRACT |
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Heat shock transcription factor 2 (HSF2) is a
transcription factor that regulates heat shock protein gene expression,
but the mechanisms regulating the function of this factor are unclear. Here we report that HSF2 is a substrate for modification by the ubiquitin-related protein SUMO-1 and that HSF2 colocalizes in cells
with SUMO-1 in nuclear granules. Staining with anti-promyelocytic leukemia antibodies indicates that these HSF2-containing nuclear granules are PML bodies. Our results identify lysine 82 as the major
site of SUMO-1 modification in HSF2, which is located in a "wing"
within the DNA-binding domain of this protein. Interestingly, SUMO-1
modification of HSF2 results in conversion of this factor to the active
DNA binding form. This is the first demonstration that SUMO-1
modification can directly alter the DNA binding ability of a
transcription factor and reveals a new mechanism by which SUMO-1
modification can regulate protein function.
In the past several years, a number of reports have described the
covalent attachment of several proteins similar to ubiquitin in their
ability to target proteins in the cell. The best studied of these is
SUMO-1, a 97-amino acid, 11-kDa polypeptide, which shares 18% amino
acid sequence identity with ubiquitin. Originally SUMO-1 was identified
as a modifier of the Ran GTPase activating protein
(RanGAP1)1 (1, 2). Unlike
ubiquitin, SUMO-1 modification of proteins does not appear to target
proteins for degradation and instead appears to have a number of other
functions in the cell (3-6). For RanGAP-1, SUMO-1 modification is
required for its association with Nup358 or RanBP2 (Ran-binding
proteins in the nuclear pore complex) and localization to the nuclear
pore complex (7, 8). SUMO-1 modification of PML is correlated with
localization of this protein to nuclear bodies, which are discrete
subdomains within the nucleus (9-13).
Like ubiquitin, SUMO-1 uses a multi enzyme ligase complex to attach to
target proteins, but the specific enzymes are distinct from those
involved in ubiquitination. The processed SUMO-1 is a substrate for the
SUMO E1 enzyme, which is a heterodimer of two proteins called
SUMO-1-activating enzyme 1 (SAE1)
and SUMO-1-activating enzyme 2 (SAE2) (14, 15). Ubc9 is the E2 for SUMO-1 conjugation, receiving the SUMO-1 from SAE1/2 and transferring it to the target protein (15, 16). All SUMO-1-modified proteins characterized to date
interact with Ubc9 directly, suggesting that SUMO-1 does not require a
separate E3 ligase for specificity (12, 17-19). Supporting this idea
is the finding that SUMO-1 modification can be reconstituted in
vitro with only ATP, SUMO-1, SAE1, SAE2, Ubc9, and the target
protein (12, 17).
Heat shock transcription factor 1 (HSF1) and heat shock transcription
factor 2 (HSF2) are transcription factors that regulate the expression
of heat shock protein (hsp) genes (20, 21). HSF1 DNA-binding is
activated in response to cell stress, but the signals and mechanisms
that regulate HSF2 function are not yet clear. In this study, we show
that HSF2 is a substrate for SUMO-1 and SUMO-2 modification in
vitro and identify lysine 82 as the primary site of SUMO-1
modification. Furthermore, we show that HSF2 colocalizes with SUMO-1 in
nuclear domain structures, and that these HSF2-containing structures
are PML bodies. SUMO modification of HSF2 results in a significant
increase in DNA binding activity of this protein. Thus, it appears that
SUMO-1 modification regulates HSF2 function by modulating the DNA
binding activity and possibly also the subcellular localization of this transcription factor.
Plasmids--
The yeast-two-hybrid vector pGBD-HSF2 was cloned
as previously described (22). Polymerase chain reaction (PCR) was used to generate BglII sites immediately before and after the
open reading frame of the mouse HSF2 Yeast Two-hybrid Analysis--
The Saccharomyces
cerevisiae strain PJ 69-4A (MATa trp 1-901 leu2-3, 112 ura3-52 ade2-101 his3-200 gal4 Immunoprecipitation Analysis--
Cells were lysed in a solution
containing 0.15 M Tris-HCl (pH 6.7), 5% SDS, and 30%
glycerol, which was then diluted 1:10 in phosphate-buffered saline
(PBS)/0.5% Nonidet P-40 plus complete protease inhibitor (Roche). Four
microliters of anti-HSF2 polyclonal antibody were added to the lysate,
incubated for 1 h at 4 °C with gentle inversion mixing, after
which protein-G-Sepharose was added. After incubation for 3 h, the
beads were collected, washed four times with ice-cold PBS, 0.5%
Nonidet P-40 plus complete protease inhibitor mixture.
Immunoprecipitated proteins were analyzed by SDS-PAGE and Western blot
using anti-SUMO-1 monoclonal antibodies (21C7) (1).
In Vitro SUMO-1 Modification Assay--
Full-length HSF2 protein
was in vitro translated in a rabbit reticulocyte lysate
system and then subjected to in vitro SUMO-1 modification
assay essentially as previously described (12).
Transient Transfection of HeLa Cells--
HeLa cells were
transfected with pEGFP-C1 or pEGFP-HSF2 Immunofluorescence Analysis--
For immunofluorescence analysis
of HeLa cells transfected with EGFP-HSF constructs, transfected cells
on coverslips were fixed with cold methanol and then blocked in cold
PBS (137 mM NaCl, 2.7 mM KCl, 8 mM
Na2HPO4, 1.5 mM
KH2PO4) +2% bovine serum albumin (BSA Fraction
V, Sigma). Nontransfected cells on coverslips were fixed using 2%
paraformaldehyde in PBS at room temperature as described previously
(24). Coverslips were then incubated for 60 min with one of the
following primary antibodies in PBS containing 2% BSA: HSF2 rat
monoclonal antibody from Neomarkers (1:100 dilution), SUMO-1 mouse
monoclonal antibody 21C7 (1:1000 dilution) (1), or PML mouse monoclonal
antibody from Santa Cruz Biotechnology, Santa Cruz, CA (1:100
dilution). After washing with PBS + 2% BSA, the coverslips were
incubated 30 min with a 1:200 dilution of the appropriate secondary
antibody linked to either the Texas Red fluorochrome or fluoroscein
isothiocyanate (Vector Laboratories, Burlingame, CA). After washing
with PBS + 2% BSA and PBS, some coverslips were also incubated 5 min
with 50 ng/ml 4',6-diamidino-2-phenylindole (DAPI) in PBS. Coverslips
were washed briefly in distilled water and mounted on a slide with
Vectashield (Vector Laboratories). Immunostaining was visualized using
a Nikon fluorescent microscope with a 60× objective and a Nikon
Spotcam digital-imaging camera.
Site-directed Mutagenesis of HSF2--
Point mutants were
generated in pcDNA-HSF2 Gel Mobility Shift Assay--
In vitro translated
HSF2 protein, with or without subsequent in vitro SUMO-1
modification, was incubated with 20 µl of binding buffer (10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol)
containing 0.1 ng of 32P end-labeled DNA probe, 0.5 µg of
poly(dI-dC)-poly(dI-dC), and 10 µg of BSA at 20 °C for 10 min. The
oligonucleotide probe contains four inverted repeats of the heat shock
element consensus sequence 5'-nGAAn-3'. After incubation, binding
reactions were subjected to electrophoresis on native 4%
polyacrylamide gels in 0.5 × TBE, and HSF2 DNA-binding complexes
were visualized by autoradiography.
Two-hybrid Analysis of the HSF2/Ubc9 Interaction--
During the
course of yeast two-hybrid analysis we discovered the existence of an
interaction between HSF2 and the Ubc9 protein. As shown in Fig.
1, yeast containing an HSF2 bait plasmid
(pGBD-HSF2) and a prey plasmid containing the region of the Ubc9
protein corresponding to amino acids 4-128 (pVP16 In Vivo and in Vitro SUMO-1 Modification of HSF2--
Interaction
with Ubc9 is a characteristic of many proteins shown
subsequently to be modified by SUMO-1. To test whether this was true
for HSF2, we immunoprecipitated HSF2 from cell extracts and then
subjected the immunoprecipitate to Western blot using SUMO-1
antibodies. The results, shown in Fig.
2A, reveal the existence of a
band of the expected size of ~80 kDa, suggesting that HSF2 is
modified by SUMO-1 in vivo.
We also tested whether the HSF2 protein is a substrate for SUMO-1
modification in vitro (Fig. 2B). This assay
contained 35S-labeled in vitro translated HSF2,
purified recombinant SUMO-1 and Ubc9, a HeLa cell extract (which
contains the SUMO E1 enzyme activity of the SAE1/2 heterodimer), ATP,
and an ATP regenerating system. When the HSF2 protein is incubated with
the HeLa extracts alone, a faint higher molecular weight protein
corresponding in size to the SUMO-1-modified form of HSF2 appears (Fig.
2B, lane 2). This is presumably because of small
amounts of endogenous SUMO-1 and Ubc9 in the HeLa extracts. However,
when SUMO-1 and Ubc9 are added to the reaction mixture a substantial
increase in amount of the higher molecular weight product is observed, corresponding to SUMO-1-modified HSF2 (lane 6). HSF2 appears
to also be a substrate for SUMO-2 modification in vitro
(lane 7).
To verify that the higher molecular weight band appearing in these
in vitro modification reactions does in fact correspond to
SUMO-1-modified HSF2, we compared the sizes of HSF2 modification products that result from the use of His6-SUMO
versus GST-SUMO-1 as the SUMO-1 substrate for the reaction.
As expected, the use of GST-SUMO-1 instead of His6-SUMO-1
results in the appearance of a larger size corresponding to the
approximate 26-kDa difference in size because of the GST moiety (Fig.
2C).
HSF2 Is Colocalized with SUMO-1 in PML Bodies--
Numerous
SUMO-1-modified proteins localize to discrete nuclear domain structures
known as PML nuclear bodies, including PML, HIPK2, and Sp100 (9-12,
18, 25). Thus, we were interested in determining whether HSF2 was
colocalized with SUMO-1 in PML nuclear bodies. To test this possibility
we performed double-label immunofluorescence analysis on HeLa cells
using antibodies that recognize HSF2 and SUMO-1. The results, shown in
Fig. 3, demonstrate colocalization
between HSF2 and SUMO-1 staining in nuclear structures in these cells.
Preabsorbing the SUMO-1 and HSF2 antibodies with purified recombinant
SUMO-1 or HSF2 protein prior to staining completely abolished the
nuclear domain structure staining that had already been observed with
these antibodies (data not shown).
As an additional test of colocalization between HSF2 and SUMO-1, we
transfected HeLa cells with a green fluorescent protein (GFP)-HSF2
fusion protein expression plasmid and then stained with the anti-SUMO-1
monoclonal antibody as well as with DAPI for visualization of the
nucleus (Fig. 4). Only a few of the cells that were transfected with pEGFP-HSF2
Next, to definitively demonstrate that the nuclear structures observed
with the HSF2 and SUMO-1 antibodies are PML nuclear bodies, we
performed double-label immunofluorescence analysis on HeLa cells using
antibodies that recognize HSF2 and PML. The results, shown in Fig.
5, demonstrate a good correlation between staining for HSF2 and PML, consistent with our hypothesis.
Identification of the SUMO-1 Modification Site in HSF2--
To
identify amino acid residues of HSF2 that are modified by SUMO-1, we
analyzed the mouse HSF2
Mutations were made in pcDNA-HSF2
We also made the K82R mutation in the HSF2-GFP mammalian expression
construct used in the experiments above to determine the effect this
had on HSF2 localization to PML bodies. Our results revealed that the
mutant HSF2-GFP still localized to nuclear bodies (data not shown). We
think this may be because of complex formation between the mutant HSF2
and endogenous wild type HSF2 present in these cells, so that the
endogenous HSF2 essentially carries the mutant HSF2 to the localization
site within PML bodies. This is consistent with our previous results
showing spontaneous HSF2 trimer formation in cells transfected with
HSF2 expression constructs (28). A similar explanation has been
suggested for results of experiments on PML SUMO mutants, in which
failure of mutant proteins to localize to PML bodies was observed only
in cells lacking endogenous PML (13, 29).
SUMO-1 Modification Activates HSF2 DNA Binding--
Lysine 82 is
located within the DNA-binding domain of HSF2, and so we hypothesized
that SUMO-1 modification at this site might regulate the DNA binding
function of this protein in some way. To test this possibility, we
compared the DNA binding activity of HSF2 that was SUMO-1-modified
in vitro to that of unmodified HSF2 using the gel shift
assay with a probe that specifically binds HSFs. Fig.
7 shows that SUMO-1 modification of wild
type HSF2 results in a significant increase in DNA binding activity of
this protein (compare lane 1 and lane 2).
However, no such increase in DNA binding activity is observed for the
HSF2 K82R mutant subjected to the SUMO-1 modification reaction,
consistent with the hypothesis that SUMO-1 modification of this residue
is responsible for this increase in DNA binding activity.
The regulation and function of HSF2 as a transcription factor are
still largely a mystery. The only thing that is really clear is that
HSF2 can bind to heat shock elements and regulate transcription of heat
shock protein genes. However, very little is known about how and under
what conditions HSF2 DNA binding and function are regulated. In
addition, in contrast to the related family member HSF1 that mediates
the stress-induced expression of hsp genes, it is not clear what
biological function HSF2 may be performing with regards to regulating
hsp expression. Adding to the complexity, recent data from our
laboratory suggest that HSF2 also has a role in regulating PP2A
activity in cells via its interaction with the PR65 subunit of PP2A
(22, 30).
The results of this study shed new light on HSF2 by demonstrating that
HSF2 is a target for modification by SUMO-1. A key finding of this
study is that SUMO-1 modification causes activation of HSF2 DNA binding
ability. This is the first demonstration that SUMO-1 modification can
directly regulate the DNA-binding ability of a transcription factor,
thus expanding our knowledge of the repertoire of mechanisms by which
this modification can regulate protein function.
Regarding the mechanism by which SUMO-1 modification activates HSF2
DNA-binding, one possibility is that the modification causes a
conformational change leading to trimerization and DNA binding ability.
Consistent with this hypothesis, lysine 82, the lysine residue of HSF2
indicated by our results to be a site for SUMO-1 modification, is
located in a so-called "wing" within the DNA-binding domain of this
protein. It is interesting to note that that this wing region
has already been suggested to play a role in stabilizing the trimeric
DNA binding form of HSF by forming interactions between monomers within
the trimer (31). Thus, SUMO-1 modification of HSF2 at this lysine
residue could induce a conformational change that makes these wings
accessible for interaction and stimulates trimer formation.
Consistent with the importance of SUMO-1 modification for HSF2
function, sequence analysis reveals that the consensus SUMO-1
modification site found at Lys-82 in mouse HSF2 is conserved in both
the human and chicken HSF2 homologs (32, 33).
SUMO-1 modification could also be involved in regulating the stability
of the HSF2 protein within cells. Several examples exist which
demonstrate that SUMO-1 modification can stabilize proteins against
degradation, such as in the case of I SUMO-1 modification of HSF2 is also correlated with its localization to
PML nuclear bodies, as has been demonstrated for a number of other
proteins including PML itself and p53 (9-13, 39). What role might HSF2
localization to PML bodies play in the regulation and function of this
transcription factor? There are several possibilities, some of which
have been proposed for other transcription factors that have been found
to reside within these bodies (40, 41). First, one function might be to
bring HSF2 into close proximity with other transcriptional co-factors
that also localize to PML bodies so it can form complexes with these
proteins that are important for the subsequent ability of HSF2 to
regulate gene expression. Another possibility is that localization of
HSF2 to these bodies serves a sequestering function, tying up this pool
of HSF2 so that it can't act to regulate gene expression until some
signal triggers its release from this body. A particularly intriguing question to address in future studies is whether the SUMO-1-induced activation of HSF2 DNA binding is unique to this transcription factor
or whether it may also play a role in regulating the DNA binding
activity of other transcription factors that are SUMO-1 modified.
Studies to test these and other hypotheses will likely increase
understanding of HSF2 function in cells as well as the biological
functions of SUMO modification and PML bodies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cDNA. The
BglII-digested PCR fragment of HSF2
was cloned into the
BamHI site of pQE9 (Qiagen), thus generating pQE9-HSF2
.
The pGEX-SUMO-1 plasmid was a generous gift of Dr. Joana Desterro (16).
PCR was used to generate a SalI site and a Kozak consensus
sequence (5'-CCACC-3') immediately before and a ClaI site
immediately after the open reading frame of the mouse HSF2
cDNA.
This undigested PCR fragment of HSF2
was cloned into the
SmaI site of the pGEM-7Z cloning vector (Promega), in which
the ClaI site had been destroyed, thus generating the plasmid pGEM-HSF2
SC. The plasmid pcDNA-HSF2
-MH6
was cloned by digesting pGEM-HSF2
SC with SalI and
HindIII to liberate the majority of the HSF2 ORF and cloning
it into pcDNA3.1/MycHisA(
) (Invitrogen) digested with
XhoI and HindIII. The remaining portion of the
HSF2
open reading frame was cloned by PCR using primers that spanned the endogenous HindIII site in HSF2 and added a
HindIII site immediately 5' to the endogenous stop codon.
The HindIII-digested PCR fragment was cloned into the
HindIII site of the previous construct, and orientation of
the insert was verified using PCR. The insert for pEGFP-HSF2
was
generated by digesting pGEM-HSF2
SC with ClaI, filling the
resulting ends in with Klenow DNA polymerase (New England Biolabs,
Beverly, MA), and digesting with SalI. The insert was then
cloned into pEGFP-C1 (CLONTECH) digested with
SalI and SmaI to create pEGFP-HSF2
.
gal80
LYS2::GAL1-HIS3 GAL2-ADE2
met2::GAL7-lacZ) were cotransformed with
yeast GAL4 DNA-binding domain fusion plasmids (bait) and VP16
activation domain fusion plasmids and plated onto yeast minimal
selective medium as previously described (23). Colonies were
transferred onto plates that contained the yeast minimal selective
medium and also onto plates that additionally lacked adenosine or
histidine, which are complemented by the two-hybrid assay
reporter gene.
independently using
LipofectAMINE 2000 (Life Technologies, Inc.). In brief, HeLa cells were
seeded on coverslips such that the cells would be ~80% confluent by
the following morning. HeLa cells were grown in DMEM, 10% fetal bovine
serum (FBS), 50 µg/ml gentamicin at 37 °C with 5%
CO2. For each transfection, 4 µg of DNA and 7.5 µl of
LipofectAMINE 2000 was used according to the manufacturer's protocol.
The DNA/LipofectAMINE mixture was incubated with HeLa cells for 6 h at 37 °C with 5% CO2. After 6 h, the
DNA-containing DMEM was removed and replaced with 3 ml of DMEM, 10%
FBS, 50 µg/ml gentamicin. The cells were analyzed 24-36 h later by
fluorescence microscopy.
-MH6 that were expected to
change the three predicted SUMO-1-modified lysine residues to arginine.
The predicted residues are Lys-82, Lys-139, and Lys-151. Site-directed
mutagenesis was performed using the QuickChange mutagenesis kit
(Stratagene) according to the manufacturer's protocol. Mutations were
confirmed by DNA sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ubc9) are
able to grow on selective media. The relevant negative control plasmid
combinations, pGBD-HSF2 + pVP16 and pGBD-C2 + pVP16
Ubc9, did not
confer growth on these selective media, indicating specificity of the
interaction. Also shown for comparison is the interaction between HSF2
and a protein called PR65, for which we have previously demonstrated a
strong and specific interaction (22) (Fig. 1).
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Fig. 1.
Two-hybrid analysis of the HSF2/Ubc9
interaction. Yeast strain PJ64-4A was transfected with the
pGBD-HSF2 + pVP16 Ubc9, pGBD-HSF2 + pVP16, and pGBD-C2+ pVP16
Ubc9
and then streaked on plates containing different selective media.
Medium lacking tryptophan (-Trp) and leucine
(-Leu) selects for both plasmids. Media lacking adenosine
(-Ade) or histidine (-His) are selective for two
reporter genes in the S. cerevisiae strain PJ69-4A. Also
shown as a positive control is the previously demonstrated interaction
between HSF2 and the PR65 protein (pGBD-HSF2 + pACT2-PR65(6B)).
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Fig. 2.
In vivo and in vitro
modification of HSF2 by SUMO-1. A, HSF2 protein
was immunoprecipitated from extracts of HeLa cells followed by Western
blot using anti-SUMO-1 antibodies. The positions of molecular weight
standards are indicated on the left side of the panel.
B, in vitro translated 35S-labeled
HSF2 protein was incubated with HeLa cytosol, Ubc9, SUMO-1, SUMO-2, or
with various combinations of each of these and then subjected to
SDS-PAGE followed by autoradiography. The positions of unmodified and
SUMO-modified HSF2 are indicated to the right of the panel.
C, in vitro translated 35S-labeled
HSF2 protein was subjected to the in vitro SUMO-1
modification assay using either His6-SUMO-1 or GST-SUMO-1
as the SUMO-1 substrate for the reaction.
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Fig. 3.
Colocalization between HSF2 and SUMO-1.
HeLa cells were subjected to immunofluorescence analysis using
antibodies against HSF2 (green channel) and SUMO-1
(red channel). Lower and higher magnification images are
shown in the upper and lower sets of panels,
respectively. The yellow nuclear-localized granules in the
merged image indicate colocalization of these two proteins in these
structures.
had the punctate nuclear GFP-HSF2 staining (~7%), with the majority of the cells displaying cytosolic staining. However, in those cells that contained GFP-HSF2 nuclear domain staining, the HSF2 nuclear domain structures did colocalize with SUMO-1.
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Fig. 4.
Colocalization between GFP-HSF2 and
SUMO-1. Transiently transfected HeLa cells expressing GFP-HSF2
were stained with an antibody against SUMO-1 and DAPI for nuclear
staining. Shown are the GFP-HSF2 (green), SUMO-1
(red), and DAPI (blue) staining from three
representative fields of cells. GFP-HSF2 and SUMO-1 colocalize
(GFP-HSF2 + SUMO-1) in discrete domains (seen as
yellow dots) within the nucleus (GFP-HSF2 + DAPI).
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Fig. 5.
Colocalization between HSF2 and PML.
HeLa cells were subjected to immunofluorescence analysis using
antibodies against HSF2 (green channel) and PML (red
channel). Lower and higher magnification images are shown in the
upper and lower sets of panels, respectively. The yellow
nuclear-localized granules in the merged image indicate colocalization
of these two proteins in these structures.
protein sequence for consensus SUMO-1
modification sites (26, 27). Most SUMO-1-modified proteins described to
date conform to a consensus modification site of (I/V/L)KX(D/E) (6). This sequence analysis revealed that
HSF2 contains three matches to this SUMO-1 consensus modification
sequence, centered around Lys-82, Lys-139, and Lys-151.
-MH6 that changed
each of these lysine residues to arginine, K82R, K139R, and K151R. We then tested the ability of each of these mutant HSF2 proteins to
undergo SUMO-1 modification relative to wild type HSF2. The results
(Fig. 6A) suggest that among
these three sites, lysine 82 is a major site of SUMO-1 modification in
HSF2, because this mutant failed to show the appearance of the major
HSF2-SUMO band, whereas mutation of Lys-139 and Lys-151 did not affect
the ability of HSF2 to undergo SUMO-1 modification in this assay. In
this experiment we also noted the existence of a faint band, the
mobility of which was slightly reduced relative to the major
HSF2-SUMO-1 band, particularly noticeable in lane 4 where
the major HSF2-SUMO band is absent. We suspect this band may arise from
the use of a minor SUMO-1 modification site. Shown in Fig.
6B is a comparison of the sequence surrounding lysine 82 of
HSF2 with characterized SUMO-1 modification sites in other
proteins.
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Fig. 6.
Lysine 82 of HSF2 is a site of SUMO-1
modification in vitro. A, in
vitro translated 35S-labeled wild type HSF2 protein
and the HSF2 SUMO-1 consensus site mutants K82R, K139R, and K151R were
used as substrates in in vitro SUMO-1 modification
reactions. The positions of unmodified and SUMO-modified HSF2 proteins
are indicated to the right of the panel. B,
alignment of modification sites in SUMO-1-modified proteins. The
asterisk indicates SUMO-1-modified lysine residue. Conserved
lysine and glutamic acid residues are indicated in shaded
columns.
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Fig. 7.
SUMO-1 modification increases HSF2 DNA
binding activity. In vitro translated HSF2 was
subjected to in vitro SUMO-1 modification and then analyzed
by gel shift assay (lane 2) using a specific HSF-binding
oligonucleotide along with unmodified HSF2 (lane 1).
The results obtained for the HSF2 K82R mutant are shown in
lane 3 and lane 4.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, Mdm2, and possibly
p53 (34-37). Measurements indicate that the HSF2 protein has a
relatively short half-life within cells, on the order of 60 min (38).
Thus, SUMO-1 modification of HSF2 could serve to render a pool of HSF2
resistant to degradation, or conversely, perhaps regulated removal of
SUMO-1 is the controlling step that regulates the timing of HSF2 turnover.
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ACKNOWLEDGEMENTS |
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We thank Dr. Joana Desterro and Dr. Ron Hay for the generous gift of the GST-SUMO-1 bacterial expression plasmid. We are also very grateful to Dr. Wally Whiteheart for expert advice on the immunofluorescence analysis.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HD32008 and American Cancer Society Grant RPG 99-217 (to K. D. S.) and National Institutes of Health Training Grant ES 07266 postdoctoral support (to Y. H.).
§ These authors contributed equally to this work.
** To whom correspondence should be addressed. Department of Biochemistry, University of Kentucky, 800 Rose Street, Lexington, KY 40536. Tel.: 859-323-5777; Fax: 859-323-1037; E-mail: kdsarge@pop.uky.edu
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M00866200
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ABBREVIATIONS |
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The abbreviations used are: RanGAP1, Ran GTPase-activating protein 1; HSF1, heat shock transcription factor 1; HSF2, heat shock transcription factor 2; PML, promyelocytic leukemia; DAPI, 4',6-diamidino-2-phenylindole; PCR, polymerase chain reaction; hsp, heat shock protein; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; GFP, green fluorescent protein; GST, glutathione S-transferase; SAE1, SUMO-1-activating enzyme 1; SAE2, SUMO-1-activating enzyme 2; BSA, bovine serum albumin; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis.
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