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J. Biol. Chem., Vol. 278, Issue 32, 30091-30097, August 8, 2003
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¶
From the
Johns Hopkins University, Bloomberg
School of Public Health, Department of Biochemistry and Molecular Biology,
Baltimore, Maryland 21205 and Immunobiology
Center, Mount Sinai School of Medicine, New York, New York 10029
Received for publication, February 6, 2003 , and in revised form, May 19, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, but not PIAS1,
functions as an E3 ligase to promote STAT1 modification. In addition, we have
mapped the major site for SUMO modification on STAT1 to lysine 703. This
lysine residue is in close proximity to the regulatory tyrosine residue at
position 701, whose phosphorylation mediates STAT1 activation in response to
cytokine signaling. Mutation of lysine 703 to arginine abolishes SUMO
modification of STAT1 both in vitro and in vivo. However,
this mutation does not affect the activation of STAT1 or the ability of either
PIAS1 or PIASx- to function as an inhibitor of STAT1-mediated
transcription activation. Our findings demonstrate that inhibition of STAT1 by
PIAS proteins does not require SUMO modification of STAT1 itself. SUMO
modification of STAT1 may nonetheless be functionally important given the
close proximity between the SUMO modification site and tyrosine 701. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The general features of STAT1 activation are fairly well characterized;
however, the mechanisms leading to STAT1 inactivation are less well defined.
Recently, a family of proteins termed "protein inhibitors of activated
STATs" (PIAS) was identified through their direct interactions with the
STAT proteins (7,
8). Functional characterization
of the PIAS proteins revealed that they inhibit the transcription activation
of STAT-regulated genes. There are currently five unique PIAS proteins (PIAS1,
PIAS3, PIASy, PIASx-, and PIASx-
), each with distinct activities
toward the different STAT family members. PIAS1 and PIAS3, for example,
specifically inhibit STAT1- and STAT3-mediated gene activation, respectively
(7,
8). The specific mechanism by
which the PIAS proteins inhibit STAT activity in vivo is unclear,
although in vitro studies suggest that they may regulate interactions
between STATs and DNA (7).
After their identification as regulators of STAT function, the PIAS proteins were shown to function as regulators of SUMO (small-ubiquitin like modifier) modification (9–15). Like ubiquitination, SUMO modification occurs through a series of enzymatic steps (16). The first step involves the ATP-dependant formation of a thioester bond between SUMO and the E1 heterodimer consisting of Aos1 and Uba2. The second step involves the transfer of SUMO to the E2-conjugating enzyme Ubc9. In the third and final step, SUMO is covalently linked to a lysine residue in the targeted substrate. Recognition of the substrate involves direct interactions between Ubc9 and the substrate (1, 17) but may also be facilitated by proteins functioning as E3-like recognition factors. PIAS proteins have been shown to enhance the SUMO modification of a growing list of proteins that includes p53 (12, 13), c-Jun (10, 13), Sp3 (11), androgen receptor (10, 18), glucocorticoid receptor (19), LEF1 (20), IRF1 (21), and C/EBP (CCAAT/enhancer-binding protein) (22), and they have therefore been proposed to function as SUMO E3-like factors.
The link between PIAS proteins and STAT has led us to hypothesize that
STAT1 is a SUMO substrate and that its ability to activate transcription may
be regulated by SUMO modification. In this paper we demonstrate that STAT1 is
modified by SUMO both in vitro and in vivo and that
PIASx- acts as an E3 ligase for STAT1 modification. We mapped the site
for SUMO modification of STAT1 to lysine 703, a residue that is in close
proximity to the regulatory tyrosine 701. Phosphorylation of tyrosine 701 in
response to cytokine signaling is essential for STAT1 dimerization and nuclear
translocation, suggesting that SUMO modification of lysine 703 may play an
important regulatory role. In vivo transcription studies, however,
indicate that SUMO modification at lysine 703 is not required for
PIAS-mediated inhibition of STAT1 transcription activation. Our results
demonstrate that the inhibition of STAT1 by PIAS proteins occurs through
alternate mechanisms.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were generous gifts from
Dr. Ke Shuai (University of California, Los Angeles, CA). pCMV-STAT1-FLAG,
pcDNA1/SUMO-3, pcDNA6/STAT1, pEGFP-C1/SUMO-1, pEGFP-C1/SUMO-2,
pGEX-4T-1/STAT1, pGEX-4T-1/PIAS1,PGEX-4T-1/PIASx-, and PGEX-4T-1/STAT1
were all produced using standard PCR and sub-cloning techniques. Site-directed
mutagenesis of STAT1 constructs was performed using the QuikChange
site-directed mutagenesis system (Stratagene, La Jolla, CA). All mutants were
verified by DNA sequence analysis.
Protein Expression and Purification—Recombinant SUMO-1,
Aos1/Uba2, and Ubc9 were expressed and purified as previously described
(1). Recombinant PIAS1 and
PIASx- were produced in bacteria by transforming with the appropriate
pGEX-4T-1 vector and inducing expression with 0.1 mM
isopropyl--D-galactopyranoside at 20 °C for 6 h.
Recombinant proteins were purified by affinity chromatography on
glutathione-Sepharose beads (Amersham Biosciences). Recombinant PIAS1 and
PIASx- were subsequently cleaved from the beads by thrombin protease as
outlined by the manufacturer (Amersham Biosciences). STAT1 was transcribed and
translated in rabbit reticulocyte lysate in the presence of
[35S]methionine as outlined by the manufacturer (Promega Corp.,
Madison, WI).
In Vitro SUMO Modification Assays—In vitro SUMO modification
of STAT1 was performed using two individual assay conditions. Assays performed
using high concentrations of Aos1/Uba2 and Ubc9 contained 0.5 µM
Aos1/Uba2, 12 µM Ubc9, 10 µM mature SUMO, 2
mM ATP, 5 mM MgCl2, 10% glycerol, 50
mM Tris, pH 7.5, and 2 µl of in vitro translated STAT1.
Assays performed using limiting concentrations of Aos1/Uba2 and Ubc9 contained
0.3 µM Aos1/Uba2, 0.3 µM Ubc9, 7.7
µM mature SUMO, 2 mM ATP, 5 mM
MgCl2, 10% glycerol, 50 mM Tris, pH 7.5, and 2 µl of
in vitro translated STAT1 or 200 ng of recombinant STAT1. Reactions
were incubated at 37 °C for 15 min. Reactions were quenched with SDS-PAGE
sample buffer and subsequently analyzed by SDS-PAGE and autoradiography or by
immunoblot analysis with a STAT1 p91 (C-24) antibody (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA).
Cell Culture, Transient Transfection, and Transcription Reporter
Assays—U3A cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. Cells were plated onto 12-well tissue
culture plates 12 h before transfection and transfected with the indicated
plasmid DNAs (300 ng of STAT1, 300 ng of SUMO, 100–1200 ng of PIAS)
using Lipofectin as described by the manufacturer (Invitrogen). Total DNA
concentrations in each transfection experiment were kept constant by adding
empty vector DNA to 1.6 µg. 3 ng of a pRL-TK Renilla luciferase
control plasmid was transfected into all cells to normalize for transfection
efficiencies. In all cases, cell lysates were analyzed or processed 24 h
post-transfection. When indicated, interferon-
(PeProTech, Rocky Hill,
NJ) (10 ng/ml) was added 6 h before luciferase assays were performed or 1 h
before immunoblot analysis. For luciferase reporter assays, cells were lysed
in 100 µl of reporter lysis buffer, and dual luciferase assays were
conducted according to the manufacturer's instructions (Promega). Protein
expression of all transfected plasmids was checked by immunoblot analysis
using appropriate antibodies (anti-GFP antibody (Clontech, Palo Alto, CA) and
anti-FLAG M2 antibody (Sigma).
Nickel Affinity Chromatography—Cells were lysed in 100 µl
of buffer containing 6 M guanidine hydrochloride, 10 mM
Tris, 100 mM NaH2PO4, 300 mM NaCl,
and 10 mM imidazole at pH 8.0 24 h post-transfection. Cell lysates
were mixed gently with 25 µl of nickel nitrilotriacetic acid-agarose
(Qiagen, Valencia, CA) for1hat room temperature. Beads were washed 3 times
with buffer containing 8 M urea, 10 mM Tris, 100
mM NaH2PO4, 300 mM NaCl, and 20
mM imidazole at pH 6.3. Bound proteins were eluted from the beads
using 40 µl of SDS-PAGE sample buffer. Immunoblot analysis was performed
using the STAT1 p91 (C-24) antibody (Santa Cruz Biotechnology).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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91-kDa
full-length STAT1 was produced as well as several smaller protein products
possibly resulting from translation initiation at internal methionines or from
proteolysis of the full-length protein
(Fig. 1, lane 1).
Translated STAT1 was added to an assay reaction containing 10 µM
SUMO, 12 µM Ubc9, and 0.5 µM Uba2/Aos1, conditions
under which most SUMO substrates can be modified in the absence of exogenously
added E3 ligases. When analyzed by SDS-PAGE, a novel protein band could be
detected migrating with a molecular mass of 106 kDa
(Fig. 1, lane 3).
Conjugation of a single molecule of SUMO to protein substrates resulted in an
15-kDa shift in molecular mass, suggesting that the 106-kDa protein
corresponds to STAT1 conjugated to SUMO at a single lysine residue. The
presence of the 106-kDa band was dependent on SUMO, because it was not
detected in reactions containing only exogenously added E1 and E2 enzymes
(Fig. 1, lane 2).
Modification reactions containing glutathione S-transferase-SUMO
produced an appropriately higher molecular mass band of 135 kDa
(Fig. 1, lane 4),
presenting further evidence that the shifted bands correspond to SUMO-modified
STAT1.
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STAT1 was modified in the above assay in the presence of relatively high
concentrations of E1 and E2 enzymes and in the absence of an exogenously added
E3 enzyme. This is consistent with the findings for most SUMO substrates,
where E3 ligases are required only when E1 and E2 enzyme concentrations are
significantly lower. To assay for the ability of PIAS proteins to function as
E3 ligases for STAT1, we therefore repeated the above assay using reduced
concentrations of Aos1/Uba2 and Ubc9. Translated STAT1 was added to an assay
containing 7.7 µM SUMO, 0.3 µM Ubc9, and 0.3
µM Uba2/Aos1. A faint protein band was detected by SDS-PAGE,
indicating that STAT1 was not efficiently modified by SUMO under these reduced
E1 and E2 enzyme conditions (Fig.
2A, lane 2). Assays using the same low
concentrations of Uba2/Aos1 and Ubc9 were also performed in the presence of
exogenously added PIAS1 or PIASx-. Surprisingly, we observed weak
activity for PIAS1 (Fig.
2A, lane 3) but found that PIASx- could
stimulate the SUMO modification of STAT1 in an E3-like manner
(Fig. 2A, lane
4). As a control for PIAS1 activity, we also performed assays using the
androgen receptor as a substrate. Both PIAS1 and PIASx- were able to
enhance the SUMO modification of the androgen receptor to the same extent as
previously shown (18,
19), indicating that both
proteins are equally active (Fig.
2B). There are two shifted bands corresponding to
SUMO-modified androgen receptor because androgen receptor is modified at two
lysines (23).
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Rabbit reticulocyte lysate contains many factors that could affect the
efficiency of SUMO modification. We therefore expressed STAT1 in bacteria as a
glutathione S-transferase fusion protein and assayed for its ability
to be modified in reactions containing only purified, recombinant factors.
Recombinant STAT1 was incubated with an assay mix containing 7.7
µM SUMO, 0.3 µM Ubc9, and 0.3 µM
Uba2/Aos1. These conditions were neither sufficient to modify in
vitro translated STAT1 efficiently
(Fig. 2A, lane
2) nor were they sufficient to modify recombinant STAT1, as observed by
immunoblot analysis (Fig. 3,
lane 2). To determine whether PIASx- could stimulate SUMO
modification of recombinant STAT1, a similar SUMO modification reaction was
supplemented with 0.3 µM PIASx-. Immunoblot analysis
revealed that the addition of PIASx- stimulated SUMO modification of
the recombinant STAT1, as revealed by the presence of a protein band of
132 kDa (15 kDa larger that unmodified glutathione
S-transferase-STAT1) (Fig.
3, lane 3). These results further demonstrate that STAT1
is modified by SUMO in vitro and indicate that PIASx-
functions directly as an E3 ligase to promote STAT1 modification.
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STAT1 Is Modified at a Single Lysine Residue at Position
703—STAT1 has a modular structure with the amino terminus
containing a coiled-coil domain, the central region containing the DNA binding
domain, and the carboxyl terminus containing an SH2 domain followed by a
transactivation domain (Fig.
4A). STAT1 contains two potential SUMO modification sites
defined by the consensus sequence 
KXE, where is a
hydrophobic residue, K is the lysine residue modified by SUMO-1, X is
any amino acid, and E is glutamic acid
(17,
24). This consensus sequence
defines the site modified in many (but not all) SUMO substrates. The consensus
sequences in STAT1 reside around lysine 110 in the amino terminus and lysine
703, which is in the carboxyl-terminal region between the SH2 domain and the
transactivation domain (Fig.
4A). The consensus sequence surrounding lysine 703 is of
particular interest because of its close proximity to tyrosine 701, the
tyrosine residue phosphorylated in response to cytokine stimulation. To
determine whether either of these sites in STAT1 is modified by SUMO, we
mutated lysine residues 110 and 703 to arginine. Mutant and wild-type STAT1
proteins were translated in rabbit reticulocyte lysate in the presence of
[35S]methionine, and their ability to be modified by SUMO was
determined using in vitro assays followed by SDS-PAGE
(Fig. 4B). Both
wild-type STAT1 and the lysine 110 to arginine mutant STAT1 could be modified
by SUMO, as determined by the appearance of the 106-kDa STAT1-SUMO
conjugate (Fig. 4B,
lanes 1–2 and 4–5). In contrast the 106-kDa
band corresponding to SUMO-modified STAT1 was not detected with the lysine 703
to arginine mutant STAT1 (Fig.
4B, lanes 7–8). These SUMO modification
assays were performed in the presence of high concentrations of E1 and E2
enzymes and no added E3, although similar results were also obtained with low
concentrations of E1 and E2 enzymes in the presence of PIASx-
(Fig. 4B, lanes 3,
6, and 9). These results indicate that lysine 703 is the major
site for SUMO modification of STAT1 in vitro. The results also
indicate that PIASx- enhances specifically the modification at lysine
703.
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STAT1 Is Constitutively Modified by SUMO in Vivo—The above
results indicate that STAT1 is modified by SUMO in vitro and
that this modification, which occurs on a lysine residue only two amino acids
away from tyrosine 701, may be functionally significant. Therefore, we next
performed experiments to assess whether STAT1 is modified by SUMO in
vivo and also whether its modification may be influenced by PIAS
overexpression and/or by cytokine stimulation. U3A cells, which do not express
endogenous STAT1, were transfected with plasmids encoding for either
Myc-His-tagged wild-type STAT1 or Myc-His-tagged lysine 703 to arginine mutant
STAT1. 24 h after transfection, cells were lysed in guanidine hydrochloride,
and STAT1 protein was purified by nickel affinity chromatography. Immunoblot
analysis of the affinity-purified proteins with an anti-STAT1 antibody
revealed unmodified STAT1 but no detectable higher molecular mass bands
corresponding to possible STAT1-SUMO conjugates
(Fig. 5, lanes 2 and
6). Overexpression of SUMO by transient transfection can enhance the
modification of many SUMO substrates in vivo. Therefore, cells were
co-transfected with an Myc-His STAT1 plasmid and plasmids encoding for
GFP-tagged SUMO-2 (we have observed that STAT1 is equally modified by SUMO-1,
SUMO-2, and SUMO-3 in our in vitro and in vivo experimental
conditions; data not shown). GFP-tagged SUMO was used because it causes a
unique shift in molecular mass of 45 kDa. Cells were again lysed 24 h
after transfection, and STAT1 was purified by nickel affinity chromatography.
Under these conditions, a faint protein band with the predicted molecular mass
of a STAT1-GFP-SUMO conjugate was detected by immunoblot analysis, but only in
cells transfected with wild-type STAT1
(Fig. 5, lane 4) and
not in cells transfected with the lysine 703 to arginine STAT1 mutant
(Fig. 5, lane 8).
These results indicate that a small fraction of STAT1 is modified by SUMO at
lysine 703 in vivo.
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Our in vitro findings indicated that PIASx- could act as an
E3 ligase and stimulate the SUMO modification of STAT1. We therefore examined
whether overexpression of PIASx- could enhance SUMO modification of
STAT1 in vivo. U3A cells were co-transfected with an Myc-His STAT1
plasmid, GFP-SUMO plasmid, and a plasmid encoding for FLAG-PIASx-.
Cells were lysed 24 h after transfection, and STAT1 was purified by nickel
affinity chromatography. Immunoblot analysis revealed a significant
enhancement of the SUMO modification of wild-type STAT1 in the presence of
overexpressed PIASx- (Fig.
5, lane 4 versus 5). No SUMO-modified STAT1 could be
detected in cells transfected with the lysine 703 to arginine STAT1 mutant
(Fig. 5, lane 9) or in
cells overexpressing only STAT1 and PIASx- but not SUMO
(Fig. 5, lanes 3 and
7). These results indicate that PIASx- stimulates the SUMO
modification of STAT1 in vivo and further demonstrate that STAT1 is
modified on lysine 703. The expression of transfected plasmids was confirmed
by immunoblot or immunofluorescence analysis
(Fig. 5B; data not
shown).
Previous studies indicate that PIAS proteins may act specifically on
activated STAT dimers (25),
suggesting that STAT1 SUMO modification may be up-regulated in response to
STAT1 activation. To determine whether STAT1 activation and dimerization had
any influence on SUMO modification, we treated cells with -interferon
(IFN-) for 30 min after transfection with plasmids encoding for STAT1
or STAT1 and Myc-tagged SUMO-3 (again we have observed that STAT1 is equally
modified by SUMO-1, SUMO-2, or SUMO-3 in vitro and in vivo).
Myc-tagged SUMO was used in this experiment to provide further evidence that
STAT1 is indeed modified by SUMO in vivo. The Myc-tagged SUMO is
expected to cause an 15-kDa shift in STAT1 molecular mass rather than an
45-kDa shift observed with GFP-SUMO. After 30 min of IFN-
stimulation, cells were lysed, and STAT1 was purified and analyzed by
immunoblot analysis as described above. STAT1-SUMO conjugates were not
detectable in cells transfected with only STAT1 either in the absence or in
the presence of IFN- (Fig.
6, lanes 3 and 4). In cells co-transfected with
both STAT1 and SUMO, STAT1-SUMO conjugates of the predicted molecular mass
were detected in both treated and untreated cells at equivalent levels
(Fig. 6, lanes 5 and
6). These results indicate that STAT1 is constitutively modified by
SUMO in unstimulated cells and that IFN- does not have any appreciable
affect on the overall levels of STAT1 SUMO modification.
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We were unable to demonstrate directly using SUMO antibodies that the
higher molecular mass forms of STAT1 are in fact SUMO conjugates. However,
collectively, the data provide compelling evidence that STAT1 is modified by
SUMO at lysine 703 in vivo. First, detection of the higher molecular
mass forms of STAT1 was enhanced in cells overexpressing SUMO. Second,
detection of the higher molecular mass forms of STAT1 was further enhanced by
co-expression of PIASx-, which we found promotes SUMO modification of
STAT1 in vitro. Third, the higher molecular mass forms of STAT1 that
were detected corresponded to the predicted molecular masses of STAT1-SUMO or
STAT1-GFP-SUMO, depending on the form of SUMO overexpressed in the cells.
Finally, the higher molecular mass forms of STAT1 were not detected in cells
expressing the lysine 703 to arginine mutant STAT1, which we showed is not
modified by SUMO in vitro.
SUMO Modification of STAT1 at Lysine 703 Is Not Required for
PIASx- or PIAS1-mediated Inhibition of
STAT1—Previous studies using in vivo reporter assays
indicate that PIAS proteins can inhibit STAT-mediated gene activation
(7,
25,
26). Our results demonstrate
that STAT1 can be modified by SUMO at lysine 703 in vivo and that
PIASx- can act as an E3 ligase to stimulate modification of STAT1 at
this residue. To determine whether SUMO modification of STAT1 at lysine 703 is
functionally related to PIAS-mediated STAT1 inactivation, luciferase assays
were preformed using a reporter construct containing a (3x)Ly-6E promoter
fused to the firefly luciferase gene. (3x)Ly-6E is a construct consisting of
three copies of a promoter element found in the murine Ly-6A/E gene, which is
transcriptionally induced by treatment of cells with IFN-, , or
(27).
We first performed experiments to determine whether the lysine 703 to
arginine mutant STAT1 could activate gene expression in a manner comparable
with wild-type STAT1. U3A cells were transiently transfected with FLAG-tagged
wild-type STAT1 or a lysine 703 to arginine mutant STAT1 and the (3x)Ly-6E
reporter construct. The transfected cells were treated with IFN- for 6
h, and cell lysates were then assayed for luciferase activity. Cells
co-transfected with wild-type STAT1 and (3x)Ly-6E yielded a 4.6-fold
increase in luciferase expression in response to IFN- compared with a
5-fold increase when the lysine 703 to arginine mutant STAT1 was
co-transfected with the reporter construct
(Fig. 7A). These
results indicate that a lysine to arginine substitution at residue 703 in
STAT1 does not inhibit STAT1 ability to activate transcription in response to
IFN- stimulation. The slight, but reproducible increase in
transcription activation observed with the lysine 703 to arginine mutant STAT1
relative to the wild-type STAT1 suggests that SUMO modification of lysine 703
may play an inhibitory role.
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We next assayed for the ability of PIAS1 and PIASx- to inhibit gene
activation induced by wild-type STAT1. It has previously been shown that PIAS1
is able to inhibit STAT1-mediated gene activation
(7,
25,
26); however, the affects of
PIASx- on STAT1 gene activation have not been definitively
characterized. When FLAG-tagged PIAS1 was co-transfected in increasing amounts
with wild-type STAT1, PIAS1 reduced IFN--stimulated transcriptional
activation in a dose-responsive manner
(Fig. 7B), similar to
that previously observed by others
(7,
25,
26). When FLAG-tagged
PIASx- was co-transfected with wild-type STAT1, a reduction in gene
activation was also observed (Fig.
7B). Both PIAS1 and PIASx- were expressed at
comparable levels in these experiments, as determined by immunoblot analysis
(data not shown). At these expression levels PIASx- acted as a more
potent inhibitor of STAT1 gene activation, reducing induced expression of the
reporter gene by 2.1-fold at the lower protein concentration compared with
1.1-fold inhibition by PIAS1.
Finally, we examined the ability of PIAS1 and PIASx- to inhibit gene
activation mediated by the lysine 703 to arginine mutant STAT1 that cannot be
modified by SUMO. Cells were co-transfected with the lysine 703 to arginine
STAT1 mutant, the (3x)Ly-6E reporter construct, and PIAS1 or PIASx-.
Cell lysates were again prepared 6 h after IFN- treatment for
luciferase assays. These assays revealed that the lysine 703 to arginine
mutant STAT1 is inhibited by both PIAS1 and PIASx-
(Fig. 7C). Most
significantly, the levels of inhibition were comparable with the levels of
inhibition observed with wild-type STAT1. These results demonstrate that the
PIAS proteins are able to inhibit interferon-induced gene activation by STAT1
in a manner that is independent of STAT1 SUMO modification.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(but not PIAS1) can function as an E3 ligase to enhance the SUMO
modification of STAT1. Overexpression of PIASx-, like PIAS1, was able
to inhibit STAT1-mediated gene activation in cultured cells. Significantly,
however, both PIAS1- and PIASx--mediated inhibition of STAT1 gene
activation was found to be independent of SUMO modification of STAT1
itself.
The specific mechanism by which PIAS proteins inhibit STAT1-activated
transcription in vivo has not previously been defined. In
vitro experiments suggest that direct interactions between PIAS1 and
STAT1 may interfere with the STAT1 ability to bind DNA
(7). The recent discoveries
that PIAS proteins function as SUMO E3 ligases, however, has raised the
possibility that PIAS-mediated inhibition of STAT1 transcription may involve
direct SUMO modification of STAT1. Although our findings indicate that STAT1
can be modified by SUMO, several lines of evidence indicate that this
modification does not play a direct role in the PIAS-mediated inhibition of
transcription measured in our assays. First, our results indicate that both
PIASx- and PIAS1 are able to inhibit STAT1-mediated gene activation
in vivo but that only PIASx- acts as a SUMO E3 ligase that
enhances the SUMO modification of STAT1. Most significantly, PIAS1 and
PIASx- were able to inhibit both wild-type STAT1 and a mutant STAT1 not
able to be modified by SUMO.
Together these findings indicate that the PIAS proteins can inhibit STAT1
through a mechanism that is not dependent on direct SUMO modification of
STAT1. Inhibition may involve interactions between STAT1 and the PIAS proteins
that prevent DNA binding and/or interaction with other regulatory factors, as
previously suggested (7).
Alternatively, it is also possible that the observed inhibition of STAT1 may
involve PIAS-mediated SUMO modification of transcription regulators other than
STAT1. Notably, overexpression of both PIAS1 and PIASx- in
vivo enhances the SUMO modification of many proteins (data not shown)
(10). Although the precise
identity of these SUMO conjugates is not known, they are likely to include
numerous transcriptional regulators known to undergo SUMO modification. It is
conceivable that the enhanced modification of some of these factors could
indirectly regulate STAT1 activity. Consistent with this model, LEF1
(20), p53
(13), C/EBP
(22), and the progesterone
receptor (15) are all
inhibited by PIAS protein overexpression, independent of their ability to be
directly modified by SUMO.
Although our findings do not reveal a relationship between SUMO modification of STAT1 and PIAS-mediated inhibition of its transcriptional activity, our findings do nonetheless establish that STAT1 is a SUMO substrate. To address the effects of SUMO modification on STAT1, more specific approaches to selectively enhance the levels of STAT1 SUMO modification in vivo are required. However, the position of modification at lysine 703 provides insights that allow us to speculate on the role for SUMO modification of STAT1. Analysis of the crystal structure of STAT1 reveals that residues encompassing the SUMO modification site are located at the dimer interface (28). Significantly, however, amino acid residues likely to be directly involved in interactions with Ubc9 and subsequent SUMO modification (isoleucine 702, lysine 703, and glutamic acid 705) are exposed on the surface of the protein dimer (Fig. 8; Ref. 28). The positions of these residues suggest that the phosphorylated STAT1 dimer could be a substrate for SUMO modification, a suggestion that is supported by preliminary in vitro studies (data not shown).
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The enzymes involved in SUMO conjugation (including the PIAS proteins) are
concentrated in the nucleus, making it reasonable to hypothesize that SUMO
modification of STAT1 is a nuclear event. SUMO modification of STAT1 dimers in
the nucleus could have a stabilizing effect by protecting the dimer from
phosphatases, or it could play a role in recruiting co-factors that regulate
STAT1-mediated transcription activation. Alternatively, SUMO modification at
lysine 703 could destabilize STAT1 dimers by directly interfering with
dimerization or by attracting the TC45 phosphatase involved in STAT1
dephosphorylation (29).
Interestingly, we found that the lysine 703 to arginine mutant STAT1 showed
modest but consistently stronger IFN- induced gene activation relative
to wild-type STAT1. This observation suggests that SUMO modification of STAT1
may repress its ability to activate gene expression. Several additional
transcription factors that are modified by SUMO behave in a similar manner
(22,
23,
30,
31).
In conclusion, we have shown that STAT1 is a SUMO substrate and that
PIASx- is able to act as an E3 ligase and enhance its SUMO
modification. Most importantly, we have demonstrated that inhibition of STAT1
by overexpression of PIAS proteins does not require direct SUMO modification
of STAT1 itself. We propose a model in which PIAS overexpression leads to the
SUMO modification of other transcription regulators that indirectly affect
STAT1-mediated gene activation. Identification of these other regulatory SUMO
substrates will be an important task for the future. In addition, it will also
be of great interest to determine how SUMO modification of STAT1 at lysine
residue 703 regulates its functions.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 410-614-6878; Fax: 410-955-2926; E-mail: mmatunis{at}jhsph.edu.
1 The abbreviations used are: STAT, signal transducers and activators of
transcription; PIAS, protein inhibitors of activated STAT; SUMO,
small-ubiquitin like modifier; E1, ubiquitin-activating enzyme; E2, ubiquitin
carrier protein; E3, ubiquitin-protein isopeptide ligase; GFP, green
fluorescent protein; IFN, interferon.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  M. A. Park, Y.-J. Seok, G. Jeong, and J.-S. Lee SUMO1 negatively regulates BRCA1-mediated transcription, via modulation of promoter occupancy Nucleic Acids Res., January 17, 2008; 36(1): 263 - 283. [Abstract] [Full Text] [PDF]
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 Md. M. Ali, T. Yoshizawa, O. Ishibashi, A. Matsuda, M. Ikegame, J. Shimomura, H. Mera, K. Nakashima, and H. Kawashima PIASxbeta is a key regulator of osterix transcriptional activity and matrix mineralization in osteoblasts J. Cell Sci., August 1, 2007; 120(15): 2565 - 2573. [Abstract] [Full Text] [PDF]
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J.-H. Man, H.-Y. Li, P.-J. Zhang, T. Zhou, K. He, X. Pan, B. Liang, A.-L. Li, J. Zhao, W.-L. Gong, et al. PIAS3 induction of PRB sumoylation represses PRB transactivation by destabilizing its retention in the nucleus Nucleic Acids Res., November 14, 2006; 34(19): 5552 - 5566. [Abstract] [Full Text] [PDF]
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 W. A. Sands, H. D. Woolson, G. R. Milne, C. Rutherford, and T. M. Palmer Exchange Protein Activated by Cyclic AMP (Epac)-Mediated Induction of Suppressor of Cytokine Signaling 3 (SOCS-3) in Vascular Endothelial Cells. Mol. Cell. Biol., September 1, 2006; 26(17): 6333 - 6346. [Abstract] [Full Text] [PDF]
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 M. Eckert, S. E. M. Meek, and K. L. Ball A Novel Repressor Domain Is Required for Maximal Growth Inhibition by the IRF-1 Tumor Suppressor J. Biol. Chem., August 11, 2006; 281(32): 23092 - 23102. [Abstract] [Full Text] [PDF]
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 K. Yamashina, H. Yamamoto, K. Chayama, K. Nakajima, and A. Kikuchi Suppression of STAT3 Activity by Duplin, Which Is a Negative Regulator of the Wnt Signal J. Biochem., February 1, 2006; 139(2): 305 - 314. [Abstract] [Full Text] [PDF]
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J.-M. Wang, C.-Y. Ko, L.-C. Chen, W.-L. Wang, and W.-C. Chang Functional role of NF-IL6{beta} and its sumoylation and acetylation modifications in promoter activation of cyclooxygenase 2 gene Nucleic Acids Res., January 5, 2006; 34(1): 217 - 231. [Abstract] [Full Text] [PDF]
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G. Forget, D. J. Gregory, and M. Olivier Proteasome-mediated Degradation of STAT1{alpha} following Infection of Macrophages with Leishmania donovani J. Biol. Chem., August 26, 2005; 280(34): 30542 - 30549. [Abstract] [Full Text] [PDF]
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D. Ungureanu, S. Vanhatupa, J. Gronholm, J. J. Palvimo, and O. Silvennoinen SUMO-1 conjugation selectively modulates STAT1-mediated gene responses Blood, July 1, 2005; 106(1): 224 - 226. [Abstract] [Full Text] [PDF]
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