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doi:10.1182/blood-2006-04-020271
Prepublished online July 20, 2006;
Li Song, Samita Bhattacharya, Ali A Yunus, Christopher D Lima and Christian Schindler
Stat1 and SUMO modification
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Song & Bhattacharya Stat1 SUMOylation
1
Stat1 and SUMO modification
Li Song*
1
, Samita Bhattacharya*
1
, Ali A. Yunus
2
, Christopher D. Lima
2
and Christian
Schindler
1,3,†
Departments of
1
Microbiology and
3
Medicine, Columbia University, New York, NY
10032 USA &
2
Structural Biology Program, Memorial Sloan-Kettering Cancer Center,
New York, NY
10021 USA
*These authors contributed equally to this study.
†
Corresponding Author
Christian Schindler
Columbia University, HHSC 1208
701 West 168th Street
New York, NY 10032
Tel. 212 305-5380
Fax 212 543-0063
e-mail cws4@columbia.edu
Grant Information
This research was supported by NIH grants AI05821
†
, GM65872
2
, the Burroughs
Wellcome Fund (APP#2010)
†
and the Rita Allen Foundation
2
.
Manuscript Information
Number of Figures: 7
Number of Tables: 1
Abstract Word Count: 188
Text Word Count: 4,320
Number of references: 44
Key Words – Cytokine / IFNs / Signal transduction / STATs / SUMO
Subject Categories – Immunobiology
Running Title – Stat1 and SUMOylation
Blood First Edition Paper, prepublished online July 20, 2006; DOI 10.1182/blood-2006-04-020271
Copyright © 2006 American Society of Hematology
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Song & Bhattacharya Stat1 SUMOylation
2
Abstract
Many proteins are known to undergo SUMO (Small Ubiquitin-related Modifiers)
modification by an E1, E2 and E3 dependent ligation process. Recognition that PIAS (Protein
Inhibitor of Activated STATs) proteins are SUMO E3 ligases raised the possibility that STATs
may also be regulated by SUMO modification. Consistent with this possibility, a SUMOylation
consensus site (ΨKxE; Ψ = hydrophobic residue, x = any residue) was identified in Stat1 (i.e.,
702
IKTE
705
), but not in other STATs. Biochemical analysis confirmed that Stat1 K
703
could be
SUMO modified in vitro. Mutation of this critical lysine (i.e., Stat1
K703R
) yielded a protein,
which when expressed in Stat1-/- MEFs, exhibited enhanced DNA binding and nuclear retention.
This was associated with modest changes in transcriptional and antiviral activity. However,
mutation of second critical residue in the SUMO consensus site, E
705
(i.e., Stat1
E705A
), yielded a
protein with wild type DNA binding, nuclear retention, transcriptional and antiviral activity.
Similar observations were made when these mutants were expressed in primary Stat1-/-
macrophages. These observations suggest that although Stat1 can uniquely be SUMOylated in
vitro, this modification is unlikely to play an important role in regulating Stat1 activity in vivo.
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3
Introduction
Characterization of the ability of type I IFNs (e.g., IFN-α) to rapidly activate genes led to
the identification of ISGF-3, a transcription factor consisting of Stat1, Stat2 and an IRF-9 DNA
binding protein
1
. Subsequently, IFN-γ was shown to induce genes through Stat1 homodimers
2
.
To date, seven STATs (Signal transducers and activators of transcription) have been identified in
vertebrates, all of which are activated by phosphorylation on a single tyrosine (Y701 in Stat1;
reviewed in 3,4). Activation drives STAT dimerization by directing a stable and specific
association between the phosphotyrosine of one STAT and the SH2 domain of a partner STAT
5
.
Residues located at positions +1, +3, +5, +6 and +7 carboxy terminal to this phosphotyrosine
(i.e., amino acids 702, 704, 706 and 707 for Stat1) determine the specificity of this interaction
6
.
Dimerized STATs translocate to the nucleus, where they bind to members of the GAS (gamma
activated site) family of enhancers, culminating in the induction genes
3,4
.
The regulation of STAT signal decay has also been an area of active investigation. Four
major classes of counter regulatory molecules have been identified, including phosphatases
3,4,7
,
nuclear “transportases”
8-10
, covalent modifiers
4,11,12
and specific STAT counter-regulatory
proteins (e.g., SOCS and PIAS proteins
13,14
). Studies on SOCS-1 have provided significant
evidence for a critical role in down regulating IFNγ-Stat1 dependent signals, but studies on PIAS
(Protein Inhibitor of Activated STATs) proteins have yielded less direct mechanistic insight into
Stat1 regulation
14-16
. More recent studies have determined that PIAS proteins are SUMO E3
ligases (see below), raising the possibility that STAT activity is regulated through SUMO
modification
17-19
.
SUMOs (Small Ubiquitin-related Modifiers) are ~100 amino acid peptides, which like
ubiquitin, become covalently attached to cellular target proteins (reviewed in 17,18,19).
However, in contrast to ubiquitin, SUMO modifications do not target proteins for degradation,
but rather promote protein-protein interactions, direct subcellular localization and/or serve to
antagonize ubiquitin dependent degradation. SUMO conjugation entails the formation of a
reversible isopeptide bond between the C-terminus of the SUMO peptide (SUMO-1, SUMO-2 or
SUMO-3) and the ε amino group of the lysine found in the consensus sequence ψKxE (ψ =
hydrophobic residue, “x” = any residue; see Table 1). Analogous to ubiquitin, SUMO
conjugation is mediated by an ATP-dependent E1 activating complex (i.e., Aos1+Uba2), an E2
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Song & Bhattacharya Stat1 SUMOylation
4
ligation complex (i.e., Ubc9) and an E3 conjugation complex. The relative specificity exhibited
by Ubc9 for some SUMO substrates is likely to account for E3 independent SUMO conjugation
observed in vitro
20,21
. Finally, isopeptidases from the SUSP/SENP family assure that SUMO
modification is reversible
18,22
.
Sequence analysis revealed two potential SUMO modification sites,
109
LKEE
112
and
702
IKTE
705
, in Stat1, but not other STATs (see Table 1). In vitro SUMO conjugation studies
determined that Stat1 is SUMO modified at lysine 703, but not lysine 110. A subsequent
functional analysis of two SUMOylation resistant Stat1 mutants, Stat1
K703R
and Stat1
E705A
,
revealed two distinct phenotypes. Stat1
K703R
exhibited enhanced DNA binding, prolonged
nuclear retention, and modest changes in the biological response to IFN-γ, as recently reported
23
.
In contrast, Stat1
E705A
exhibited wild type DNA binding, nuclear retention and biological
response to IFN-γ. These observations suggest that lysine 703, located at the critical interface
between Stat1 homodimers
6
, plays a fortuitous and structurally important role in Stat1 DNA
binding activity; and that Stat1 activity is not likely to be significantly regulated by SUMO
conjugation in vivo.
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Song & Bhattacharya Stat1 SUMOylation
5
Results
Stat1 can be SUMO modified
Recent studies identifying PIAS (Protein Inhibitors of Activated STATs) proteins as
SUMO E3 ligases suggested that STATs may also be SUMO modified
17-19
. Analysis of all
mammalian STAT sequences identified two potential SUMO modification sites, Stat1 residues
109
LKEE
112
and
702
IKTE
705
(see Table 1). Efforts to exploit SUMO-1 specific antibodies to
detect endogenous SUMO-modified Stat1 were unsuccessful. To improve the chances of
detecting this product, Ubc9 and HA-SUMO-1 were over expressed in HEK-293T cells. Under
Figure 1. Stat1 SUMOylation in HEK-293T cells.
(A) HEK-293T cells were transfected with HA-SUMO-1, Ubc9 and/or PIASy. 48 hrs later they
were stimulated with IFN-γ (3 ng/ml; 30 min.). WCEs were immunoprecipitated with an anti-
Stat1 antibody and sequentially immunoblotted with HA and Stat1 antibodies. (B) (Left panel)
48 h after transfection with HA-SUMO-1 and Ubc9, HEK-293T cells were stimulated with IFN-
γ (3 ng/ml; 30 min.). WCEs were either precipitated with a biotinylated GAS oligonucleotide
(Oligo; see Table 1S) or Stat1 specific antibody (anti-Stat1). Precipitates were fractionated by
SDS-PAGE, and sequentially immunoblotted with Stat1-phosphotyrosine and Stat1 specific
antibodies. (Right panel) WCEs were prepared 48 h after transfection with His-SUMO-1 and
Ubc9, HEK-293T cells were stimulated with IFN-γ (3 ng/ml; 30 min.) and either precipitated
with a Stat1 specific antibody (anti-Stat1) or ProBond nickel beads, and sequentially
immunoblotted with Stat1-phosphotyrosine and Stat1 specific antibodies. Nonspecific (NS)
band is indicated. Data are representative of three independent studies.
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Song & Bhattacharya Stat1 SUMOylation
6
these conditions SUMO-Stat1 conjugates were readily recovered in Stat1 immunoprecipitates
(Fig. 1, panel A), as previously reported
24,25
. Co-expression of PIAS-y, however, impeded
recovery of SUMO-Stat1 conjugates, most likely because of reduced levels of SUMO-1 and
Ubc9 expressed in triple transfectants.
One potential SUMO modification site, lysine 703 (K703), lies adjacent to the tyrosine
that is phosphorylated during Stat1 activation, (i.e., Y701). It was therefore important to
determine whether SUMO conjugation affected Stat1 phosphorylation and subsequent DNA
binding activity. As anticipated, phosphorylated Stat1 was readily recovered, either by
immunoprecipitation or oligonucleotide precipitation from extracts prepared of IFN-γ stimulated
HEK-293T cells over expressing Ubc9 and HA-SUMO-1 (Fig. 1B, left panel). Although slower
migrating SUMO-Stat1 conjugates were readily recovered in the Stat1 immunoprecipitates, they
were not detected in the oligonucleotide precipitates, suggesting that SUMO modified Stat1 will
not bind DNA. Likewise, phosphorylated Stat1 was readily recovered in Stat1
immunoprecipitates prepared from IFN-γ stimulated HEK-293T cells over expressing Ubc9 and
His-SUMO-1. Yet, even though Stat1 was readily collected by nickel–His-SUMO-1 pull down
and phospho-Stat1 was abundant, no phospho-Stat1 was recovered in the nickel pulldowns (Fig.
1B, right panel). These observations suggest that Stat1 modification by tyrosine phosphorylation
and SUMOylation are mutually exclusive.
Stat1 is SUMO modified at Lysine 703
To develop more direct evidence for Stat1 SUMO modification and to map the
SUMOylation site, we turned to an effective in vitro conjugation assay that overcomes
limitations imposed by SUMO deconjugating enzymes
20,22,26
. Purified recombinant Stat1 was
incubated with purified active preparations of human recombinant SUMO-1, E1 (Uba2/Aos1),
E2 (Ubc9), and E3 (Nup358 or PIAS1)
20,21,27
. After one hour, the products were fractionated by
SDS-PAGE and immunoblotted with a Stat1 specific antibody. As shown in Figure 2, the
anticipated ~ 105 kDa SUMO-Stat1 conjugate was readily formed in an ATP dependent, but not
E3 dependent manner (panel A). Analogous results were obtained with a purified p53 control
(data not shown). The SUMO-Stat1 conjugates (~3-4 % of the total Stat1) and the remaining
non-reacted (i.e., native) Stat1 were then excised, digested with trypsin and evaluated by mass
spectrometry. The specific loss of peptides spanning K703, but not K110 indicated that Stat1
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Song & Bhattacharya Stat1 SUMOylation
7
had been SUMO modified at K703 (data not shown). To confirm that K703 was the
SUMOylation site, a Stat1 peptide spanning residues 697-711 (i.e., KGTGYIKTELISVS) was
evaluated in an in vitro SUMO conjugation system where a defective E2 (i.e., Ubc9
C93S
) served
as a negative control
28
. Several additional peptides were evaluated. This included the
analogous peptide from Stat3 (i.e., AAPYLKTKFICVT) and a Stat1 peptide in which K703 was
mutated to arginine (i.e., KGTGYIR
TELISVS). To explore whether Stat1 Y701
phosphorylation precludes SUMO modification, as suggested by HEK-293T over expression
studies (Fig. 1), a Stat1 phospho-peptide (i.e., phospho-Y701) was also evaluated. Again, p53
peptides spanning a well characterized SUMOylation site served as important controls
20
. After
5 and 24 hours of SUMO conjugation, only the wild type Stat1 peptide was SUMOylated as
effectively as the control wild type p53 peptide (see Fig. 2B). More detailed kinetic studies
provided further evidence that wild type Stat1 was an effective SUMO substrate, whereas Stat3,
phospho-Stat1 and the mutant peptides were poor substrates (Fig. 2C). These data confirmed
Figure 2. In vitro SUMO conjugation assay.
(A) Purified recombinant Stat1 (0.5 μM) is SUMOylated in vitro after incubation with SUMO-1
(2 μM), E1 (Uba2/Aos1; 0.3 μM), E2 (Ubc9; 0.3 μM) and E3 (PIAS1 or Nup358 at 0.3 μM) for
1 h at 37 °C. Samples were fractionated by SDS-PAGE and immunoblotted with anti-Stat1.
Mobility of molecular weight markers and Stat1 isoforms are indicated. (B) Wild type and
mutant p53, Stat1, PO
4
-Stat1, and Stat3 peptides (500 μM) were SUMOylated, as in panel A, in
the absence of E3, with either wild type or mutant Ubc9 for 5 or 24 h at 37 °C. Samples were
fractionated by SDS-PAGE and stained with Coomassie Blue. (C) Graphical representation of
a more detailed kinetic SUMOylation assay of peptides from panel B (t = 0, 10, 40, 60, 90 160
and 300 min). 1.0 represents maximal conjugation. Products were detected by staining the gel
with Sypro (Bio-Rad). The images were quantified on Quantity One Software (Bio-Rad).
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Song & Bhattacharya Stat1 SUMOylation
8
K703 as the SUMO modification site, but only when the adjacent Y701 was not phosphorylated.
The failure to SUMOylate the Stat3 peptide was consistent with our inability to modify purified
preparations of recombinant Stat3
9
(data not shown), reflecting a critical divergence in the
corresponding potential Stat3 SUMO conjugation site (see Table 1).
Stat1
K703R
exhibits enhanced DNA binding activity
To explore the potential role of Stat1 SUMO modification in vivo, a SUMOylation
resistant Stat1
K703R
mutant was generated. Retroviral vectors directed expression of Stat1
K703R
and wild type Stat1 at physiological levels in Stat1-/- MEFs (Murine Embryonic Fibroblasts
29
)
in > 95 % of infected cells (data not shown; see also Figs. 4A and B). Upon brief (i.e., 0.5 h)
stimulation with IFN-γ, both Stat1 and Stat1
K703R
were rapidly activated (data not shown; see
also Fig. 4B). However, when these extracts were evaluated by electrophoretic mobility shift
assay (EMSA), the DNA binding activity of Stat1
K703R
was significantly more prolonged than
that of wild type Stat1 (data not shown; see also Fig. 4C). To determine whether Stat1
K703R
Table 1. Comparison of SUMO modification consensus sites.
A
ψKxE
Stat1 107-SCLKEERKI
Stat1 700-GY
IKTELIS
Stat2 693-GY
VPSVFIP
Stat3 704-PY
LKTKFIC
Stat4 688-KYLKHKLIV
B
p53 320-HKKLMFKTEGPDSD
p53
K386M
320-HKKLMFMTEGPDSD
C
Stat1 697-KGTGYIKTELISVS
Stat1
K703R
697-KGTGYIRTELISVS
P
PO4-Stat1 697-KGTGYIKTELISVS
Stat3 701-SAAPYLKTKFICVT
(A) Potential SUMO consensus conjugation sites (ψKxE) found in Stat1, Stat2,
Stat3 and Stat4. (B) Wild type and mutant SUMO conjugation site from p53 and
p53
K386M
. (C) Wild type and mutant Stat1 SUMO consensus sites. Also shown is
the Stat1 tyrosine phosphorylation site (Y
701
).
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Song & Bhattacharya Stat1 SUMOylation
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exhibited an enhanced affinity for GAS elements, DNA dissociation studies (i.e., “off-rate”)
were performed. Stat1
K703R
displayed a four-fold slower off-rate than wild type Stat1 (i.e., t
1/2
>
120 vs. 30 minutes; see Figs. 3A and B). Likewise, Stat1
K703R
exhibited an increased relative
GAS binding activity (~ 4 fold) compared to wild type Stat1 (see Figs. 3C and D). As
anticipated, no differences were observed in ISGF3-ISRE DNA binding activity in IFN-α
stimulated extracts, where IRF-9 mediates DNA binding (data not shown). In sum, these studies
demonstrate that mutation of lysine 703 to arginine significantly enhanced Stat1 GAS binding
activity in response to IFN-γ stimulation, recently also reported in human U3A cells
23
.
Analysis of additional Stat1 SUMOylation defective mutant
To develop additional evidence that Stat1 was SUMO modified at K703 in vivo, a second
critical amino acid in the Stat1 SUMO consensus modification site (i.e., E705), which is also not
involved in dimerization, was mutated to alanine
6,17,18
. Again, retroviral vectors directed a
physiological expression of Stat1
K703R
, Stat1
E705A
and wild type Stat1 in > 95 % of Stat1-/- MEFs
Figure 3. Kinetics of IFN-γ dependent Stat1 and Stat1
K703R
DNA binding.
(A) Stat1-/- MEFs, infected with empty vector (pMIG) or retroviral vectors directing
expression of either Stat1 (St1) or Stat1
K703R
(St1
K703R
), were stimulated with IFN-γ (66 U/ml,
30 min). Dissociation kinetics of Stat1 DNA binding activity were determined by incubating
the IFN-γ stimulated extracts with a labeled GAS probe (0.1 pmol; 20 min at 22 °C) and then
chasing with an 100-fold excess of cold probe. Aliquots were removed at indicated times and
loaded onto a running gel. (B) Quantification of DNA binding from panel A was determined
by imagequant and plotted as fraction bound vs. time. (C) Relative affinity was determined
by simultaneously incubating IFN-γ stimulated WCEs (from panel A) with a labeled GAS
probe (0.1 pmol) and increasing molar excess of cold probe, as indicated (20 min, 22 °C).
Samples were then run on EMSA gel. (D) Quantification of DNA binding results from panel
C was determined by imagequant and plotted as fraction bound vs. molar excess of cold
probe. Data are representative of three independent studies.
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Song & Bhattacharya Stat1 SUMOylation
10
(Fig. 4A, top panel). To confirm that both mutants were defective in SUMO modification, HA-
SUMO-1 and Ubc9 were over expressed in these MEFs. Despite modest transfection efficiency
in these MEFs (see Fig. 4A, bottom panel), the studies clearly demonstrate the formation of the
~105 kDa SUMO-Stat1 conjugate in cells expressing wild type Stat1, but not in those cells
expressing the SUMOylation defective mutants, Stat1
K703R
and Stat1
E705A
(Fig. 4A, top panel).
Next, the IFN-γ dependent activation of Stat1
E705A
was evaluated. Stat1
E705A
exhibited a
pattern of activation (i.e., tyrosine phosphorylation) and DNA binding that was identical to that
Figure 4. Kinetics of IFN-γ dependent of Stat1, Stat1
K703R
and Stat1
E705A
activation.
(A) Stat1-/- MEFs, infected with an empty vector (pMIG), or retroviral vectors directing
expression of either Stat1 (St1), Stat1
k703R
(St1
K703R
), or Stat1
E705A
(St1
E705A
), were
subsequently transfected with HA-SUMO1 and Ubc9 cDNA constructs. Whole cell extracts
were prepared, immunoprecipitated with anti-Stat1, fractionated on 7% SDS-PAGE and
immunoblotted for Stat1 (top panel); or extracts were directly fractionated on 12% SDS-
PAGE and sequentially immunoblotted with anti-Stat1 and anti-HA. WCE from 293T cells
transfected with HA-SUMO1 and Ubc9 from Fig. 1 served as positive controls. (B) Stat1-/-
MEFs from panel A were stimulated with IFN-γ (50 U/ml; 0.5-12 h). WCEs were prepared,
fractionated by SDS-PAGE and sequentially immunoblotted with antibodies specific for
phosphotyrosine-Stat1 (top panel) and total Stat1 (bottom panel). (C) Extracts from panel B
were evaluated by EMSA with a GAS probe. Data are representative of three independent
studies in MEFs and macrophages.
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Song & Bhattacharya Stat1 SUMOylation
11
of wild type Stat1, with continuous (i.e., up to 12 h) IFN-γ stimulation (see Figs. 4B & 4C).
Likewise Stat1
K703R
exhibited an essentially normal pattern of tyrosine phosphorylation, notable
for a slight delay at early time points and more robust activity at later time points (Fig. 4B).
However, these modest changes in IFN-γ dependent Stat1
K703R
tyrosine phosphorylation
correlated with a striking increase in DNA binding activity (Fig. 4C). Thus, SUMOylation
defective Stat1
E705A
exhibits an activation profile that is distinct from Stat1
K703R
, but similar to
wild type Stat1.
The rapid and transient translocation of Stat1 to the nucleus is another characteristic
feature of Stat1 signaling activity. Since several studies have implicated SUMO modification in
the regulation of nuclear trafficking
26,30,31
, a set of Stat1 immunolocalization studies were
carried out. Analogous to the pattern observed with wild type Stat1, both Stat1
K703R
and
Stat1
E705A
exhibited a predominately cytoplasmic distribution in unstimulated cells and robust
nuclear accumulation with IFN-γ stimulation (i.e., 0.5 h; see Fig. 5). Six hours post stimulation
both Stat1 and Stat1
E705A
were fully re-exported back to the cytoplasm, rendering those cells
ready for another round of stimulation. In contrast, Stat1
K703R
continued to exhibit a strong
nuclear retention, consistent with its enhanced DNA binding activity
10
. These studies
Figure 5. IFN-γ dependent nuclear localization of Stat1, Stat1
K703R
and Stat1
E705A
.
Stat1-/- MEFs expressing physiological levels of either Stat1 (St1), Stat1
K703R
(St1
K703R
) or
Stat1
E705A
(St1
E705A
), as in Figure 4, were stimulated with IFN-γ (50 U/ml; 0, 0.5, and 6 h).
Cells were fixed and either imaged for the expression of a GFP reporter gene or after
immuno-staining with anti-Stat1. Cells were examined under a 40-x Nikon epifluorescence
objective. Data are representative of more than three independent studies.
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Song & Bhattacharya Stat1 SUMOylation
12
demonstrate that the two SUMOylation defective Stat1 mutants exhibit remarkably divergent
phenotypes.
Evaluation of the biological response mediated by Stat1
E705A
and Stat1
K703R
in MEFs
Next, several studies were undertaken to explore the potential role of SUMOylation in
regulating biological responses directed by Stat1. In the first set of studies, the ability of Stat1-/-
MEFs expressing wild type Stat1, Stat1
K703R
or Stat1
E705A
to direct the IFN-γ dependent
induction of several target genes was explored (Fig. 6). Wild type Stat1 and Stat1
E705A
promoted
similar “normal” patterns of IRF1, TAP1, GBP1 and Mig expression
32,33
. However, cells
expressing Stat1
K703R
exhibited a delayed expression pattern, with significant reduction of target
Figure 6.
IFN-γ dependent expression of target genes in Stat1, Stat1
K703R
and Stat1
E705A
MEFs.
(A) Total RNA was prepared from Stat1-/- MEFs ectopically expressing empty vector
(pMIG), Stat1 (St1), Stat1
K703R
(St1
K703R
) or Stat1
E705A
(St1
E705A
), as in Figure 4, after
stimulated with IFN-γ (50 U/ml; 0-12 h), as indicated. The expression of target genes (IRF1,
TAP1, GBP1 and Mig) was determined by Q-PCR from cDNA templates. The relative
expression of each gene was normalized to β-actin expression. (B) Stat1-/- MEFs, ectopically
expressing empty vector (pMIG), Stat1 (St1), Stat1
K703R
(St1
K703R
) or Stat1
E705A
(St1
E705A
),
were transiently transfected with a GAS driven luciferase reporter (B2SH-WT3-Luc) in
triplicate and stimulated with IFN-γ (50 U/ml; 6 h). Samples were harvested and evaluated
for luciferase and renilla activity (in arbitrary light units). Data are representative of two
independent experiments. (C) Immunoblot demonstrates that Stat1 expression (wild type and
mutants
)
was similar in all three Stat1 ex
p
ressin
g
lines.
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Song & Bhattacharya Stat1 SUMOylation
13
gene expression at 2 and 6 hours post stimulation, followed by normalization of expression by 12
hours (Fig. 6A). Changes in the expression of a GAS-driven reporter gene, which tends to
measure a more averaged transcriptional response, did not reveal any statistically significant
differences between wild type, Stat1
E705A
or Stat1
K703R
(Fig. 6B; note panel C illustrates that all
Stat1s were expressed at equivalent levels). There was however a consistent trend towards
diminished reporter expression in the Stat1
K703R
cells. Thus, Stat1
E705A
is functionally
indistinguishable from wild type Stat1, but the transcriptional kinetics in Stat1
K703R
cells are
notable for a reduced initial expression of target genes, in the setting of a mutant with enhanced
DNA binding activity.
IFNs stimulate a potent and physiologically important antiviral response, which
represents an integrated response of numerous target genes
4,34
. To determine whether
SUMOylation defective Stat1 mutants direct an abnormal antiviral response to IFN-γ, Stat1-/-
MEFs expressing either wild type Stat1, Stat1
K703R
or Stat1
E705A
were infected with Vesicular
Stomatitis Virus (VSV). In the first study, cells were pretreated with increasing doses of IFN-γ
(0.5-50 U/ml) prior to infection with a fixed multiplicity of infection (MOI = 0.5; Fig. 7A, top
panel). As anticipated, Stat1-/- cells were not protected by IFN-γ pretreatment
29,34
, but
Stat1
E705A
and wild type Stat1 directed the same robust antiviral response to IFN-γ (at 5 and 50
U/ml). Stat1
K703R
demonstrated modestly enhanced antiviral activity, but only at low IFN-γ
doses. Similarly, Stat1
K703R
directed a modestly enhanced antiviral response to IFN-γ (5 U/ml)
when the VSV MOI was varied from 0.001 to 1.0 (Fig. 7A, bottom panel). Thus, SUMOylation
defective Stat1
E705A
directs a wild type antiviral response to IFN-γ. In contrast, Stat1
K703R
, with
its enhanced DNA binding ability, directs a modestly enhanced response.
Evaluation of the biological response mediated by Stat1
E705A
and Stat1
K703R
in macrophages
Macrophages are an important physiological target of IFN-γ
4,35
. To evaluate the
biological activity of the SUMOylation defective Stat1 mutants, primary Stat1-/- bone marrow
macrophages (BMMs) were infected with retroviruses encoding wild type Stat1, Stat1
K703R
or
Stat1
E705A
. Modest differences in the level of expression were noted that correlated with an
infection efficiency, which ranged between 25-35% (not shown). First, these macrophages were
assessed for a Stat1 dependent ability to induce NO production upon stimulation with IFN-γ plus
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Song & Bhattacharya Stat1 SUMOylation
14
LPS (Fig. 7B, top panel)
34-36
. Modest differences between the capacity of wild type and mutant
Stat1 to direct NO production appeared correlated with differences in the level of Stat1
expression. Next, the Stat1 dependent ability of IFN-γ to induce MHC II expression in Stat1
“transduced” (i.e., GFP
+
) macrophages was evaluated by FACS (Fig. 7B, bottom panel)
34
.
Figure 7. Biological response of Stat1, Stat1
K703R
and Stat1
E705A
in MEFs and
macrophages.
(A) IFN-γ dependent antiviral response of Stat1, Stat1
K703R
and Stat1
E705A
MEFs. (Top
Panel) Stat1-/- MEFs ectopically expressing empty vector (pMIG), Stat1 (St1), Stat1
K703R
(St1
K703R
) or Stat1
E705A
(St1
E705A
), as in Figure 4, were infected with VSV (MOI = 0.5) after
IFN-γ (0, 0.5, 5 and 50 U/ml; 16 h) pretreatment. Viral yield in supernatants of infected cells
was determined, in triplicated, by plaque assay on Vero cells. Data is presented as total
recovered PFU (Plaque Forming Units). (Bottom Panel) Viral yield from pMIG, St1, St1
K703R
, St1
E705A
MEFs infected in triplicate with VSV at varying MOIs (0.001, 0.01, 0.1 and 1)
after IFN-γ (5 U/ml; 16 h) pretreatment. Viral titer was determined as above and is
representative of 3 independent studies. (B) IFN-γ dependent Stat1, Stat1
K703R
& Stat1
E705A
activity in macrophages. (Top Panel) Stat1-/- macrophages, infected with empty vector
(pMIG), Stat1 (St1), Stat1
K703R
(St1
K703R
) or Stat1
E705A
(St1
E705A
), were evaluated for their
capacity to produce NO 72 hrs after stimulation with IFN-γ (50 U/ml) and/or LPS (2 μg/ml).
(Lower Left Panel) Surface MHC-II expression in GFP
+
BMMs was determined by FACS,
48 hrs after stimulation with IFN-γ (50 U/ml). (Lower Right Panel) Immunoblot
demonstrates that Stat1 ex
p
ression was similar in each set of transfectants. Data are
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Song & Bhattacharya Stat1 SUMOylation
15
Again, no significant differences were observed between wild type Stat1 and the mutants,
suggesting that Stat1
K703R
and Stat1
E705A
direct wild type patterns of IFN-γ dependent responses
in primary macrophages.
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Song & Bhattacharya Stat1 SUMOylation
16
Discussion
PIAS proteins were initially identified for their capacity to regulate STATs under
conditions of over expression
14
. However, recent studies identifying PIAS proteins as SUMO
E3 ligase(s) raised the possibility that STATs may be regulated through SUMO modification
19,26,30,31,37,38
. When STAT sequences were scanned for the SUMO consensus modification site,
only two potential sties were identified in Stat1, but not in other STATs (see Table 1).
Biochemical studies identified lysine 703, adjacent to the activation tyrosine (i.e., Y701), as the
SUMO conjugation site. However, the inability to recover tyrosine phosphorylated SUMO-Stat1
conjugates in the HEK-293T cells raised concern over whether a single Stat1 molecule could be
simultaneously modified at both Y701 and K703 (Fig. 1). Consistent with this, a tyrosine
phosphorylated Stat1 peptide was a poor substrate for in vitro SUMO modification (Fig. 2).
To evaluate the potential physiological significance of SUMO Stat1 conjugation,
SUMOylation defective Stat1 mutants were generated. Fortuitously, the two most critical
residues in the SUMO consensus modification site, K703 and E705, are exposed, suggesting they
could be mutated without perturbing Stat1 activity
6,24
. Consistent with this, expression of the
first mutant, Stat1
K703R
, in Stat1-/- MEFs revealed a wild type pattern of rapid IFN-γ dependent
activation. However, the activation of Stat1
K703R
was associated with significantly enhanced
DNA binding and a prolonged pattern of nuclear retention. This observation raised the
possibility that Stat1 SUMO modification might normally serve to promote Stat1 signal decay
(e.g., by driving DNA dissociation and thereby promoting dephosphorylation). Yet, biochemical
studies failed to demonstrate that tyrosine phosphorylated Stat1, the predicted SUMOylation
target, could be SUMO modified. A second finding that was difficult to reconcile with this
model was that the whole population of SUMOylation defective Stat1
K703R
exhibited
dramatically enhanced DNA binding activity, even though only minute quantities of Stat1-
SUMO conjugates were detected in wild type cells, even under conditions of over expression
(see Figs. 1)
23-25
. The most significant data to undermine the notion that SUMO conjugation
regulates Stat1 activity came from the characterization of the second SUMOylation defective
mutant, Stat1
E705A
. This mutant was essentially indistinguishable from wild type Stat1. It
exhibited a wild type pattern of activation, signal decay, DNA binding and nuclear retention.
More importantly, in vivo studies demonstrated that Stat1
E705A
and wild type Stat1 were
equivalent in their ability to direct the expression of IFN-γ target genes and IFN-γ dependent
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Song & Bhattacharya Stat1 SUMOylation
17
antiviral responses. Similar results were obtained when Stat1
E705A
was expressed in primary
Stat1-/- macrophages. In sum, these observations provide strong evidence that SUMO
modification does not play an important role in the regulation of Stat1 activity, either in
fibroblasts or macrophages.
Upon completing our initial characterization of the Stat1 SUMOylation site, two other
groups reported K703 as a Stat1 SUMO modification site
24,25
. Both found that Stat1
K703R
exhibited relatively modest changes in Stat1
K703R
dependent transcriptional activity in primate
cells. Subsequently, one of these groups confirmed our observation of enhanced IFN-γ
dependent DNA binding activity and nuclear retention with Stat1
K703R
23
. Analogous to our own
observations, this correlated with a modest increase in the Stat1
K703R
dependent transcription of a
reporter, as well as prolonged transcription of three endogenous genes (e.g., GBP1, IRF1 and
TAP1). A second SUMOylation defective mutant, Stat1
E705A
, was also evaluated and found to
drive a modestly enhanced IFN-γ dependent expression of a GAS driven reporter gene (versus
our finding of a wild type response). However, companion DNA binding and nuclear retention
studies on Stat1
E705A
were not provided. We speculate that the modest differences between these
published studies and ours reside in the cell type employed. Our study employed Stat1 deficient
primary cells or their derivatives, and not tumor cells that had undergone extensive mutagenesis,
as is the case with U3A cells
39
. Moreover, our study employed a pool of stably “transduced”
cells, rather than a limited number clonally selected cell lines. (Of note, in our hands the pattern
of gene expression varied considerably amongst U3A clones; data not shown.) Finally, our
studies demonstrated that IFN-γ dependent biological responses, including antiviral activity, NO
production and target gene expression, were not significantly perturbed in SUMOylation
defective Stat1 mutants.
Although the unusual properties of Stat1
K703R
could easily be exploited to argue that Stat1
activity is regulated by SUMO conjugation of K703, we believe that our data provide compelling
evidence to the contrary. Notably, even under idealized reaction conditions, with purified Stat1,
only a modest fraction of Stat1 was SUMO modified (Fig. 2). This contrasted the fully penetrant
enhanced DNA binding activity of Stat1
K703R
, suggesting that this mutation causes a structural
perturbation to Stat1 dimer that stabilizes DNA binding. Consistent with this, K703 residue lies
in a critical location of the Stat1-Stat1 dimerization interface. More significantly, a second
SUMOylation defective Stat1 mutant fails to exhibit this phenotype, providing compelling
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Song & Bhattacharya Stat1 SUMOylation
18
evidence that the enhanced DNA binding is unrelated to a potential loss in the ability to be
SUMO modified. Surprisingly however, the enhanced DNA binding activity of Stat1
K703R
correlated with relatively meek changes in biological response, including a modestly enhanced
antiviral activity at the lowest doses of IFN-γ and a lack of differences in NO production. The
enhanced antiviral activity suggests that increased DNA binding activity may compensate for
low levels of activation (i.e., when active Stat1:Stat1 homodimers are rate limiting). Yet, at
more standard doses of IFN-γ this advantage is lost. The lack of correlation between the
enhanced DNA binding activity of Stat1
K703R
and target gene expression was surprising. This
may suggest that Stat1 plays a more important role in initiating transcription (i.e., an “on
switch”) than in regulating the duration of a transcriptional response
40
. Moreover, the K703R
mutation may impede recruitment of transcriptional cofactors, yielding an initially delayed
response in target gene expression. Additional point mutations of K703 in Stat1, and closely
related Stat3, will be important in exploring these possibilities.
Materials and methods
Cell culture: HEK-293T, Vero and L929 cells were from ATCC (Manassas, Virginia, USA);
and Stat1-/- mouse embryonic fibroblasts (MEFs) and macrophages were harvested from Stat1-/-
mice (generously provided by D. Levy, New York University). Cells were cultured in
Dulbecco’s Modified Eagle’s medium, supplemented with 10% fetal bovine serum from GIBCO
Laboratories (Grand Island, NY). Bone marrow (from mouse femurs) derived macrophages
were cultured in 20 % L929 cell conditioned media for 5-10 days
32
. After 16 h in culture,
adherent bone marrow cells were infected (three times) with retrovirus freshly prepared from
HEK-293T transfectants (below). Stat1-/- MEFs were infected twice with pMIG retroviruses
encoding Stat1, Stat1
K703R
and Stat1
E705A
in the presence of polybrene (8 μg/ml; Sigma, St.
Louis, MO), as previously reported
41
. High titer viral supernatants were prepared through
transient transfection in HEK-293T cells by calcium phosphate precipitation
9
. Retroviral
infection efficiency, as determined by FACS (GFP
+
cells; FACS-Calibur; BD Biosciences, San
Jose, CA), varied between 90-95 % in MEFs and 25-35 % in BMMs.
For viral response assays, MEFs were infected with Vesicular Stomatitis Virus (VSV,
Indiana strain; gift from R. Pine, Public Health Research Institute, NY) prepared in Vero cells.
Viral yield was measured 24 hrs after infection by titering on Vero cells over-layed with 1.5 %
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Song & Bhattacharya Stat1 SUMOylation
19
methyl cellulose for 36 hrs
34
. Expression of the reporter GFP gene and MHC II (after staining
with anti-mouse I-A
b
; BD Pharmingen, San Diego, CA) was evaluated by flow cytometry
(FACS-Calibur; BD Biosciences, San Jose, CA)
9,34
. NO production was evaluated at day 10 of
culture and 72 hrs after IFN-γ (50 U/ml) and/or LPS (2 μg/ml) stimulation, as previously
described
35
.
Biochemical studies: Recombinant SUMO-1, E1, and E2 were expressed, purified and assayed
as previously described
20
. The IR1-M-IR2 domains of Nup358 (aa 2596-2836) and full-length
PIAS1 (aa 1-651) were cloned and expressed as Smt3 fusion proteins, cleaved by Ulp1, and
purified by anion exchange and gel filtration chromatography
42
. To assay for Stat1
SUMOylation, purified recombinant Stat1 (0.5 μM) was incubated with SUMO-1 (2 μM), E1
(Uba2/Aos1, 0.3 μM), E2 (Ubc9, 0.3 μM) and E3 (PIAS1 or Nup358 at 0.3 μM) in a buffer with
5 mM MgCl
2
, 20 mM Hepes/pH 7.5, 1 mM DTT, 2 mM ATP and pyrophosphatase (0.5 U;
Sigma, St. Louis, MO) for 1 h at 37 °C. To assay for peptide SUMOylation, p53 (residues 323-
393), p53
K386M
(residues 320-393) Stat1/phospho-Stat1 (residues 697-711) or Stat3 (residues
701-714) peptides (at 500 μM) were incubated with E1 and either wild type or mutant Ubc9 for 5
or 24 h at 37 °C, as previously described
28
. Samples were fractionated by SDS-PAGE and
stained with Coomassie Blue or Sypro (4 - 12 h staining, > 1 h destaining; Bio-Rad, Hercules,
CA). Sypro-stained gels were imaged by UV illumination and band intensity quantified (Bio-
Rad UV system, Quantity One Software).
Over expression assays entailed transfecting pcDNA (In vitrogen, Carlsbad, CA)
expression plasmids encoding HA-SUMO-1, His-SUMO-1, Ubc9 and/or PIASy (gifts from S.
Goff, Columbia University) into HEK-293T cells by calcium phosphate precipitation or MEFs
with LipofectAmine
TM
Plus regent (In vitrogen, Carlsbad, CA). For oligonucleotide “pulldown”
assays 20 μg of a GAS oligonucleotide (see Table 1S) coupled to 75 μl biotinylated agarose
beads (Sigma, St Louis, MO) was incubated with 400 μl whole cell extracts (WCEs) for 2 h at 4
o
C, after blocking the beads with 1% BSA for 1 h at 4
o
C, as previously reported
43
. For
immunoprecipitation, 400 μl of WCEs was incubated with primary antibody for 2-16 h followed
by protein A agarose (Sigma, St. Louis, MO), and then collected, as previously reported
9,34
.
WCE or precipitates were fractionated by SDS-PAGE and then evaluated by immunoblotting
with the appropriate antibody
9
. For Nickel pulldown, 800 μl of WCEs was incubated with 100
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Song & Bhattacharya Stat1 SUMOylation
20
μl of ProBond nickel beads, washed and eluted as per manufacturer (In vitrogen, Carlsbad, CA).
For electrophoretic mobility shift assay (EMSA) extracts were incubated with a GAS probe and
fractionated by native PAGE as previously described
9,34
. Antibodies were directed against
Stat1
1
, phosphotyrosine Stat1 (Cell Signaling Technology Inc., Beverly, MA), MHC-II (BD
Pharmingen, San Diego, CA), HA (Covance, Berkeley, CA), and β-actin (Sigma, St Louis, MO).
Plasmid Constructs: pClEco and pMIG were provided by J. Luban (Columbia University)
41
.
Murine Stat1, Stat1
K703R
and Stat1
E705A
were cloned into the Xho I site in pMIG. Stat1
K703R
and
Stat1
E705A
were prepared by site directed mutagenesis (Quickchange kit, Stratagene, La Jolla,
CA) and confirmed by sequencing (see Table 1S for primer sequences).
Immunofluorescence: Cells were cultured on sterile cover slips till 20–25% confluent, fixed in
formaldehyde, and stained as previously reported
9
,
with Stat1 specific antibodies (1:250 fold
dilution) and a Cy3-conjugated secondary antibody (1:500; Jackson ImmunoResearch
Laboratories Inc., West Grove, PA). Slides were examined under a Nikon Eclipse TE300
microscope after excitation at 550 nm (Cy3) and excitation at 495 nm (GFP).
RNA expression analysis: Total RNA was prepared from MEFs with Trizol (Invitrogen
Carlsbad, CA) extraction. 4 μg of RNA were treated with RQ1 DNAse (Promega, Madison, WI)
and then reverse transcribed with SuperScript™ II (Invitrogen, Carlsbad, CA). The cDNA was
quantitatively amplified in an ABI Prism 7700 PCR system, with a SYBR green master mix
(Applied Biosystems, Foster City, CA), as previously described
32
. Gene expression was
normalized to a β-actin control.
Luciferase reporter assay: MEFs were seeded in 24 well plates (7.5 x 10
5
/well) and transfected
with 400 ng of an IRF driven luciferase reporter (i.e., B2WT3
44
) and 40 ng of pRL-tk Renilla in
LipofectAmine plus (Invitrogen, Carlsbad, CA). 24 hrs later transfectants were stimulated with
IFN-γ (5 U/ml, 6 hrs), resuspended in a passive lysis buffer, and evaluated in a luminometer (TD
20/20 Turner Systems, Sunnyvale, CA), as previously reported
44
. Experiments were done in
triplicate and luciferase activity was normalized to renilla activity.
Acknowlegements
We would like to thank Dr. Jutta Braunstein for purified preparations of recombinant
Stat1 and Stat3, and Dr. Steve Goff for reagents and advice.
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Song & Bhattacharya Stat1 SUMOylation
21
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