From the Terry Fox Molecular Oncology Group, Lady
Davis Institute for Medical Research and the Departments of
§§ Microbiology & Immunology and
§ Medicine, McGill University,
Montreal, Quebec H3T 1E2, Canada
Received for publication, September 25, 2002, and in revised form, January 9, 2003
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ABSTRACT |
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The ubiquitously expressed latent interferon
regulatory factor (IRF) 3 transcription factor is activated in response
to virus infection by phosphorylation events that target a cluster of
Ser/Thr residues,
382GGASSLENTVDLHISNSHPLSLTSDQY408
at the C-terminal end of the protein. To delineate the minimal phosphoacceptor sites required for IRF-3 activation, several point mutations were generated and tested for transactivation potential and
cAMP-response element-binding protein-binding protein/p300 coactivator association. Expression of the IRF-3 S396D mutant alone was sufficient to induce type I IFN Recognition of invading pathogens such as viruses and bacteria by
host cells is known to trigger the activation of multiple latent
transcription factors, such as NF- IRF-3 is part of the IRF family of transcription factors that includes
nine members with distinct roles in host defense against pathogens,
immunomodulation, and growth control (for review see Refs. 3, 7, and
8). Previous studies have demonstrated that the C-terminal region of
IRF-3, which comprises a cluster of phosphoacceptor sites,
382GGASSLENTVDLHISNSHPLSLTSDQY408,
is phosphorylated as a consequence of virus infection (9, 10).
Radioactive orthophosphate labeling, peptide mapping, phosphoamino acid
analysis, and decreased mobility in SDS-PAGE have shown that IRF-3 is a
phosphoprotein that is further inducibly phosphorylated upon virus
infection (10-15). The generation of point mutated forms of IRF-3
suggests that Ser385 and Ser386 (9), as well as
Ser396, Ser398, Ser402,
Ser405, and Thr404 (10) are phosphorylated
following virus infection and are involved in IRF-3 activation. Indeed,
C-terminal phosphorylation is thought to produce a change in protein
conformation that reveals the IRF association domain and the
DNA-binding domain, thus promoting dimerization and binding to
IRF-3 5'-GAAA(C/G)(C/G)GAAN(T/C)-3' consensus DNA-binding site (9, 10,
16). In addition, IRF-3 C-terminal phosphorylation is required for
association with the histone acetyltransferase nuclear proteins CBP and
p300 (9, 10, 16) causing IRF-3, which normally shuttles into and out of
the nucleus, to become predominantly nuclear (9, 10, 17). The activated
form of IRF-3, bound to CBP, induces transcription through distinct
positive regulatory domains (PRD) in the type I IFN promoters and
through select ISRE sites found in other genes such as the chemokine
RANTES, the cytokine interleukin-15, and IFN-stimulated gene (ISG) 56 (10-12, 15, 18-22). Finally, phosphorylation of IRF-3 is thought to
induce its degradation by a proteasome-mediated mechanism (10, 23).
In addition to virus infection, other stimuli such as LPS and poly(I-C)
have been shown to induce IRF-3 activation (9, 12, 15, 24-28).
However, no phosphorylation of IRF-3 in response to poly(I-C) treatment
has been demonstrated. In this case, IRF-3 activation has been observed
through nuclear accumulation, DNA binding activity, coactivator
association, and gene induction (9, 12, 15, 24). On the other hand,
phosphorylation of IRF-3 in response to LPS was demonstrated (26) but
not sufficiently to detail the precise phosphoacceptor sites.
The in vivo signaling pathways leading to IRF-3
phosphorylation and activation as well as the precise phosphoacceptor
sites remain to be elucidated. Previous studies have demonstrated
inducible N-terminal phosphorylation of IRF-3 following the activation
of mitogen-activated protein kinase kinase kinase pathways (13). More
recently, the activation of DNA-PK following virus infection was shown
to induce Thr135 phosphorylation (29). However, no
convincing physiological roles have been ascribed to these covalent
modifications. In the present study, we characterized the minimal
phosphoacceptor site(s) involved in the in vivo activation
of IRF-3 following treatment with known inducers. Of the seven
potential phosphoacceptor sites present in the C-terminal cluster, a
single point mutation of Ser396 to Asp (S396D) was
sufficient to generate a strong constitutively active form of IRF-3.
Moreover, by using a novel phosphospecific antibody, we show for the
first time that Ser396 is phosphorylated in vivo
following virus infection, nucleocapsid (N) expression or dsRNA treatment.
Reagents--
LPS (L-2654) was purchased from Sigma and
dissolved in distilled water. Poly(I-C) was purchased from Amersham
Biosciences and resuspended in phosphate-buffered saline. Sendai virus
(SeV) was a generous gift of Dr. Ilkka Julkunen (Public Health Research Institute, Helsinki, Finland).
Plasmid Constructions and Mutagenesis--
wtIRF-3, wtIRF-3 5A,
wtIRF-3 5D, wtIRF-3 3D, wtIRF-3 J2A, and wtIRF-3 J2D pFLAG constructs
and the luciferase reporter plasmids IFN Cell Culture--
Human embryonic kidney (HEK) 293 cells were
grown in Transfections, Luciferase Assays, Treatments, and
Infection--
All of the transfections were carried out on
subconfluent HEK 293 cells (calcium phosphate coprecipitation method)
or HEC-1B cells (FuGENE method) grown in 60-mm Petri dishes or 24-well
plates (for the luciferase assay). 5 µg of DNA constructs (per 60-mm dish) or 10 ng of pRLTK reporter (Renilla luciferase for
internal control), 100 ng of pGL3 reporter (firefly luciferase,
experimental reporter), and 250 ng of pFLAG expression plasmids
(24-well plate) were introduced into HEK 293 cells. At 36 h, the
cells were collected, washed in ice-cold phosphate-buffered saline, and
assayed for reporter gene activities (Promega). Infections with SeV as
well as treatments with poly(I-C) and LPS were accomplished in
serum-free medium for the first 2 h, after which 10% fetal bovine
serum was added for the rest of the incubation period. Whole cell
extracts (WCE) were prepared in Nonidet P-40 lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40,
5 mM EDTA, 10% glycerol, 30 mM NaF, 1.0 mM Na3VO4, 40 mM
Reverse Transcriptase-PCR--
Total RNA was harvested using
Trizol reagent (Invitrogen) as recommended by the manufacturer. RNA was
reverse transcribed with Superscript (Invitrogen), and the resulting
cDNA were used in a PCR with the primers described for the original
cloning of virus N cDNAs (see above) at an annealing temperature of
60 °C using Taq DNA polymerase (Amersham Biosciences).
Peptide and Antibody Production--
To generate the polyclonal
antibody specific for IRF-3 phosphorylated at Ser396,
termed HIS5033, a phosphopeptide corresponding to residues 388-402 of
human IRF-3,
KENTVDLHIS(PO Immunoblot Analysis--
To analyze the state of IRF-3
phosphorylation and to confirm the expression of the transgenes, WCE
(30-60 µg) were subjected to electrophoresis on 7.5% or 10%
acrylamide gels. The proteins were electrophoretically transferred to
Hybond-C nitrocellulose membranes (Nycomed Amersham, Inc.) in 25 mM Tris, 192 mM glycine, and 20% methanol. The
membranes were blocked in Tris-buffered saline containing 5% nonfat
dry milk and 0.1% Tween 20 for 1 h at 25 °C before incubation
for 2 h at 25 °C with anti-IRF-3 (Santa Cruz; SC-9082),
anti-FLAG M2 (Sigma), anti-I IRF-3 S396D Phosphomimetic Is a Strong Transactivator of PRD
I-III- and ISRE-containing Promoters--
To delineate the minimal
residues required for IRF-3 activation, the effects of various
phosphomimetic point mutations (Fig. 1A) on the transactivating
potential of IRF-3 were analyzed using reporter gene assays with the
IRF-3-responsive promoters IFN The Minimal Phosphoacceptor Site Required for IRF-3 Association
with CBP Coactivator Maps to Ser396--
Formation of the
IRF-3 holocomplex, which consists of an IRF-3 dimer and CBP or p300
coactivators, is a critical step in the activation of the transcription
factor (32). Association with CBP/p300 is strictly localized to the
nucleus and tethers IRF-3 in the nucleus after induction with viruses
(16, 17, 32). Therefore, the relationship between the different IRF-3
point mutants and CBP association was evaluated using
coimmunoprecipitation assays. As shown in Fig.
2, infection with SeV stimulated wtIRF-3 and CBP association (Fig. 2, A and B, lanes
1 and 2). Mutation of Ser396 to Ala
completely abrogated virus-induced CBP association (Fig. 2A,
lanes 3 and 4), whereas the phosphomimetic point
mutant S396D constitutively associated with CBP (Fig. 2B,
compare lanes 1 and 3), an association that was
enhanced following virus infection (Fig. 2B, lane
4). Similar results were obtained with the other mutants IRF-3 2A
(Fig. 2A, lanes 7 and 8) and IRF-3 5A
(Fig. 2A, lanes 13 and 14), where no
inducible association with CBP was observed. Conversely, strong
constitutive binding to CBP was present with IRF-3 2D (Fig.
2B, lane 7) and IRF-3 5D (Fig. 2B,
lane 13). As a control for these results, the S398A form of
IRF-3 was still able to associate with CBP following virus infection
(Fig. 2A, lanes 5 and 6), but only a
basal association of the phosphomimetic counterpart IRF-3 S398D with
CBP was observed in the absence of infection (Fig. 2B,
compare lanes 1 and 5). As previously reported (16), coactivator association was not observed with either the IRF-3
J2A or J2D mutants (Fig. 2, A and B, lanes
15 and 16). As discussed previously (30),
Ser385/386 phosphorylation may be required for the
sequential phosphorylation of the Ser/Thr cluster at amino acids
396-405. Together these data indicate that Ser396 is a
critical phosphoacceptor site for coactivator CBP/p300 association and
that a clear correlation exists between the capacity of the point
mutants to induce promoter activity and to associate with the CBP
coactivator.
IRF-3 S396D Expression Induces the Endogenous Interferon-stimulated
Gene, ISG56--
Recent studies have demonstrated that the
561 gene encoding the ISG56 protein is strongly induced in
response to virus infection, type I IFN, dsRNA (33) or direct
expression of IRF-3 5D (20). The effect of IRF-3 on 561 gene
induction is direct, because the promoter contains two tandem ISRE
sites, mutation of which results in the complete loss of promoter
activation by IRF-3 5D (20). To determine the effect of transient
expression of the different phosphomimetic IRF-3 forms on the induction
of the endogenous ISG56 in the IFN-unresponsive HEC-1B cells, induction
of ISG56 was analyzed by immunoblot (Fig.
3). Sendai virus infection resulted in
25-fold induction of ISG56 (Fig. 3, lane 10), whereas
wtIRF-3 expression alone resulted in a weak induction of ISG56 protein of 2.6-fold (Fig. 3, lane 2). Transfection of S396D as well
as 2D and 5D resulted in a stronger induction of the protein of 5.2-, 6.4-, and 10-fold, respectively (Fig. 3, lanes 3,
5, and 8). However, only weak inductions between
2.2- and 3.5-fold were observed with the other point mutants (Fig. 3,
lanes 4, 6, 7, and 9).
Thus, mutation at position 396 is sufficient to create a phosphomimetic form of IRF-3 that allows stable binding to CBP/p300 coactivator, transactivation of reporter genes controlled by PRD and ISRE response elements, and, importantly, enhancement of endogenous expression of
ISG56.
In Vivo Phosphorylation of Ser396 of IRF-3 following
Virus Infection--
To verify whether in vivo
phosphorylation of IRF-3 occurred at Ser396, an antibody
directed against a phosphopeptide spanning Ser396, named
HIS5033, was raised (see "Materials and Methods"). Fig. 4A shows that the antibody
reacted only toward the transfected FLAG-wtIRF-3 transgene when cells
were infected with SeV; indeed, no signal was observed in IRF-3
S396A-overexpressing cells infected with SeV. In contrast, Fig.
4B shows a specific endogenous signal when extracts derived
from SeV-infected HEK 293 cells were analyzed by immunoblot using the
phosphospecific 396-P antibody HIS5033 (Fig. 4B, lanes
2 and 3). Reblotting of the stripped membrane with an
anti-IRF-3 antibody (SC-9082) showed that the specific signal observed
with HIS5033 antibody corresponded to the slowly migrating form of
IRF-3, previously shown to represent the activated form of IRF-3 (Fig.
4B, compare lanes 4-6) (10, 13). Although IRF-3
degradation had already reduced the amount of IRF-3 in these cells at
6 h post-infection (Fig. 4B, lane 5) and the
IRF-3 signal was difficult to detect with anti-IRF-3 antibody
(SC-9082), the phosphospecific antibody HIS5033 reacted strongly under
these conditions (Fig. 4B, compare lanes 2 and 5 and lanes 3 and 6).
To investigate whether the phosphorylation of Ser396 could
be mimicked by expression of viral N protein (30), transfection experiments were performed with constructs expressing measles virus and
SeV N for 36 h post-transfection. As shown in Fig. 4C, expression of either SeV or measles virus N induced higher migrating forms of IRF-3 (middle panel). Use of HIS5033 antibody
revealed that Ser396 was phosphorylated under these
conditions (Fig. 4C, top panel). Taken together,
these results demonstrate that virus infection or expression of viral
nucleocapsid protein is sufficient to induce Ser396 phosphorylation.
IRF-3 Ser396 Is Targeted by Virus and dsRNA in
U373/CD14--
To study the kinetics of Ser396
phosphorylation in response to virus, dsRNA, or LPS, the LPS-sensitive
human U373/CD14 astrocytoma cell line was used as previously described
(34). Detailed kinetics of SeV, LPS, and poly(I-C) induction was
performed, and the phosphorylation state of Ser396 was
analyzed by immunoblot using the phosphospecific antibody HIS5033. As
shown in Fig. 5 (A and
B), both SeV and poly(I-C) induced Ser396
phosphorylation of IRF-3 with maximum signals detected at 4 and 2 h, respectively (Fig. 5, A, lanes 4-7, and
B, lanes 3 and 4). Specific
phosphoserine 396 signal was detected when IRF-3 first displayed a
retarded mobility in SDS-PAGE, as observed by the use of the SC-9082
antibody (Fig. 5, A, lanes 12-15, and
B, lanes 11-13). However, no signal was detected
when cells were treated with LPS (Fig. 5C, lanes
1-8). Furthermore, qualitative and temporal differences in the
kinetics of IRF-3 Ser396 phosphorylation by SeV and
poly(I-C) were identified: 1) Although phosphorylation of IRF-3 in
response to SeV infection, first detected at 4 h post-infection,
was followed by degradation (Fig. 5A, lanes 12-16), phosphorylation in response to poly(I-C) was detected at
2 h and did not lead to degradation. Rather, IRF-3 appeared to be
dephosphorylated over time and returned to basal forms (Fig. 5B, lanes 12-16). 2) Although Ser396
phosphorylation was not detected after LPS stimulation, a transient shift from form I to form II (Fig. 5C, lanes
13-15) was observed. This transition was previously shown to
correlate with an N-terminal phosphorylation possibly mediated through
a mitogen-activated protein kinase kinase kinase-related pathway (13).
To verify that LPS treatment was efficient, phosphorylation of
Ser32 of I IRF-3 Ser396 Phosphorylation Is Not Detected in
LPS-treated U937 Cells--
To further analyze the LPS effect, IRF-3
Ser396 phosphorylation was also examined in the monocytic
cell line U937. Fig. 6A
demonstrates that infection of U937 cells with SeV resulted in IRF-3
Ser396 phosphorylation beginning as early as 2 h
post-infection, whereas no signal with HIS5033 antibody was detected
with cell extracts derived from LPS-stimulated cells (Fig.
6B). Both SeV and LPS were able to induce the
phosphorylation of I The rate-limiting step in the activation of IRF-3 is its
phosphorylation by at least one unidentified kinase, namely
virus-activated kinase, activated following virus infection or dsRNA
treatment (9, 10, 12-15, 24). The major phosphoacceptor sites
described to date are clustered in the C-terminal end of the protein.
Yoneyama et al. (9) identified two sites, Ser385
and Ser386, in which substitution to alanine residues
completely abrogates inducible phosphorylation as diagnosed by mobility
change on SDS-PAGE, nuclear localization, association with p300
coactivator, and gene transactivation. On the other hand, our group
identified five other phosphoacceptor sites, namely Ser396,
Ser398, Ser402, Thr404, and
Ser405 (10). Mutation of these sites to aspartic acid
generates a strong constitutive active form of IRF-3, IRF-3 5D, in
which slow migration in SDS-PAGE, dimerization, CBP association, DNA
binding, and gene transactivation occur without the need of virus
infection. Conversely, generation of an IRF-3 mutant in which the five
phosphoacceptor sites are replaced with alanine completely inhibits
virus-induced dimerization, coactivator association, and
transactivation activity (10, 13, 16, 18, 21).
Despite these molecular observations, the contribution of different
phosphorylation sites has not been addressed in vivo. In a
previous study (21), we showed that the IRF-3 2A mutant behaves like
the IRF-3 5A mutant. In fact, the IRF-3 2A mutant is not phosphorylated
in response to virus infection. Moreover, as determined through
immunofluorescence analysis, the IRF-3 2A-GFP protein does not
translocate to the nucleus. These results point out the importance of
these two residues in the IRF-3 activation process. In the present
study, we demonstrate that mutation of Ser396 to Asp is the
minimal mutation required to produce a strong phosphomimetic form of
IRF-3. This observation was confirmed using a phosphospecific antibody
raised against a peptide containing the phosphorylated Ser residue at
position 396. We show for the first time that this site is
phosphorylated in vivo when cells are exposed to SeV, nucleocapsid, or poly(I-C), well characterized inducers of IRF-3. Together these results demonstrate that the Ser396 residue
is critical for IRF-3 activation.
Recent studies also suggested LPS as another IRF-3 inducer
(25-28). Indeed, treatment of murine macrophages with LPS was shown to
induce the production of IFN, suggesting the activation of some IRF
members (5, 6). In this context, Navarro and David (25) reported that
LPS treatment of human U373 astrocytoma cells resulted in IRF-3
activation (nuclear translocation and DNA binding activity) via a TLR-
and p38-dependent pathway. This observation was supported
by recent findings demonstrating that IRF-3 mediates a
TLR-3/TLR-4-specific antiviral gene program when murine B cells are
exposed to dsRNA and LPS, respectively (35). In addition, it has
recently been shown through gene disruption targeting studies that
neither the universal adaptor protein MyD88 nor the novel adaptor
TIRAP/Mal (36) were involved in IRF-3 activation following LPS
stimulation of peritoneal macrophages (26, 37). The adaptor protein
involved in LPS-induced IRF-3 activation has yet to be identified.
However, the newly identified adaptor protein TRIF appears to be
involved in dsRNA-induced IRF-3 activation by TLR-3 (Ref. 38 and
references therein), which might also be the case for IRF-3 activation
by TLR-4. Our data suggest that LPS-induced activation of IRF-3 does
not lead to phosphorylation of Ser396. How LPS activates
IRF-3 remains to be determined; treatment of target cells with LPS
generates multiple signaling pathways in addition to IKK/JNK/p38, such
as protein kinase C, Src-type tyrosine kinases, and the
phosphatidylinositol 3-kinase-protein kinase B pathway (1).
Phosphorylation of IRF-3 in response to LPS was shown by slower
mobility in one-dimensional immunoblot (26). However, the analysis of
IRF-3 phosphorylation was not resolved sufficiently to delineate the
different IRF-3 phosphorylated forms. As described previously, stress
inducers induce N-terminal IRF-3 phosphorylation, which is
characterized by a shift from form I to form II in immunoblot analysis
(13), an effect also observed with LPS in the U373 astrocytoma cell
line (Fig. 5C, lanes 13 and 14) and
B16 melanoma cells.2
Therefore, N-terminal phosphorylation or C-terminal phosphorylation at
other Ser or Thr residues may be induced by LPS and therefore involve
other signaling pathways in the activation of IRF-3. However, a limited
sensitivity of the phosphospecific antibody cannot be ruled out, and a
weak but significant phosphorylation of IRF-3 at Ser396
might occur following TLR-4 activation.
Based on the capacity of bacterially produced IRF-3 to bind the ISRE
in vitro (22, 39), it was concluded that mutation of
Ser396 to Asp in recombinant IRF-3 eliminated DNA binding
(39). This observation is in apparent contradiction with the results of
the present study demonstrating that ectopically expressed IRF-3 S396D acts as a strong transactivator of ISRE containing promoters (Figs. 1
and 3). However, a recent study demonstrated that CBP/p300 was required
for the DNA binding activity of the holocomplex (32). This observation
may explain why no stable binding to DNA was observed with recombinant
IRF-3 S396D in the absence of coactivators (39) and strengthens the
observation that IRF-3 S396D is a strong IRF-3 phosphomimetic in part
because of its capacity to bind CBP (Fig. 2).
Another surprising finding was the relative stability of the
phosphomimetic point mutants (data not shown). Indeed, whereas inactive
IRF-3 is very stable (40), virus infection results in a rapid
degradation of IRF-3 via a proteasome-dependent pathway (10, 13, 23). Based on the observation that virus-induced degradation
of IRF-3 always follows phosphorylation (10, 13), we assumed that the
destabilization of IRF-3 might be the consequence of phosphorylation of
residues in the C-terminal cluster. Based on two observations, this is
unlikely to be the signal for degradation of IRF-3 in vivo:
1) introduction of phosphomimetic point mutations did not lead to
increased degradation of IRF-3 when the mutants were overexpressed in
HEK 293 cells (data not shown) and 2) treatment of U373/CD14 with
poly(I-C) clearly induced IRF-3 Ser396 phosphorylation
(Fig. 5B, lane 4). However, this phosphorylation, which induces a shift in the migration profile of IRF-3, was not followed by degradation (Fig. 5B, lanes 11-16).
IRF-3 instability may therefore be specific to a product of the virus
life cycle that recognizes activated IRF-3 and targets it for
degradation by the proteasome pathway. Finally, the critical role of
Ser396 phosphorylation in IRF-3 activation following virus
infection and the development of a specific and sensitive
phosphospecific antibody against Ser396 may be useful as a
research and diagnostic tool as a marker of virus infection.
, IFN
1, RANTES, and the
interferon-stimulated gene 561 promoters. Using SDS-PAGE and immunoblotting with a novel phosphospecific antibody, we show for the
first time that, in vivo, IRF-3 is phosphorylated on
Ser396 following Sendai virus infection, expression of
viral nucleocapsid, and double-stranded RNA treatment. These results
demonstrate that Ser396 within the C-terminal Ser/Thr
cluster is targeted in vivo for phosphorylation following
virus infection and plays an essential role in IRF-3 activation. The
inability of the phosphospecific antibody to detect Ser396
phosphorylation in lipopolysaccharide-treated cells suggests that other
major pathways may be involved in IRF-3 activation following Toll-like
receptor 4 stimulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B, AP-1 (ATF-2/c-Jun), and the
interferon regulatory factors
(IRFs)1 (1-3). Once
activated, these transcription factors regulate the expression of a set
of genes encoding for immunomodulatory cytokines and chemokines that
are involved in the establishment of the antiviral/bacterial state,
which limits the spread of infection through innate and adaptive immune
mechanisms (1-3). The best characterized component of the innate host
defense to virus is the family of transcriptionally activated
interferon (IFN) proteins, which include type I IFN-
and IFN-
and
type II IFN-
. Type I IFNs are mainly induced in response to
infection by various types of RNA and DNA viruses (2, 4), although the
bacterial endotoxin lipopolysaccharide (LPS) induces production of IFN
in certain cells, albeit at low levels (5, 6). Once produced, these secreted proteins act in a paracrine fashion to induce gene expression in target cells in the adjacent microenvironment through engagement of
cell surface IFN receptors and the JAK-STAT signaling pathway. The
ISGF3 complex (ISGF3
/IRF-9-STAT1-STAT2) binds to
interferon-stimulated response elements (ISRE) found in numerous
IFN-induced genes, such as 2'-5' oligoadenylate synthase and the
double-stranded (ds) RNA-activated kinase, resulting in the
induction of proteins that impair viral gene expression and replication
(2, 4).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
promoter (IFNA1 pGL-3),
IFN
promoter (IFNB pGL-3), and the RANTES promoter (RANTES pGL-3)
were described previously (13, 18, 21). The expression constructs
encoding different IRF-3 C-terminal point mutants pFLAG-IRF-3 3A, 2D,
2A, S396A, S396D, S398A, S398D, S398A/S402A, and S398D/S402D were
generated by overlap PCR mutagenesis using Vent DNA polymerase. The
pCMV-2 measles N expression construct has been described previously
(30). SeV N cDNA was subcloned from pGEM SeV N plasmid (a
gift from Dr. Illka Julkunen) using 5'-AATGGCTGGGTTGTTGAGCACCTTC-3' and
5'-TTAGATTCCTCCCATCCCAGCTGCT-3' as forward and reverse primers,
respectively. The PCR-generated fragment was subcloned into pCMV-2.
-minimum essential medium. HEC-1B cells and the astrocytoma
cell line U373 overexpressing the Toll-like receptor (TLR)-4 coreceptor CD14 (U373/CD14), a gift from Dr. Michael David (University of California, San Diego, CA), were cultured in Dulbecco's modified Eagle's medium. The media were supplemented with 10% fetal bovine serum and antibiotics. U937 cells were grown in RPMI supplemented with
5% fetal clone (Hyclone) and antibiotics.
-glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride,
and 5 µg/ml of each leupeptin, pepstatin, and aprotinin) and stored
at
80 °C.
B
(MAD 10) (1 µg/ml), anti-MYC
(9E10) (1 µg/ml), or anti-ISG56 (a gift from Dr. G. Sen (Cleveland,
OH) (1:1000) in blocking solution. For the phosphospecific antibodies
anti-I
B
Ser32 phosphospecific antibody (1:2000) (New
England Biolab) and HIS5033 (1:10000), the membranes were incubated in
blocking solution for 1 h at 25 °C followed by incubation in
Tris-buffered saline containing 5% bovine serum albumin and 0.1%
Tween 20 for overnight at 4 °C. After washing four times in
Tris-buffered saline, 0.1% Tween 20, the membranes were incubated for
1 h with horseradish peroxidase-conjugated goat anti-rabbit or
anti-mouse IgG (1:10000) in blocking solution. Immunoreactive bands
were visualized by enhanced chemiluminescence (PerkinElmer Life
Sciences). For coimmunoprecipitation studies, WCE (500 µg) were
incubated with 1 µg of anti-CBP antibody A-22 (Santa Cruz) or 1 µg
of anti-FLAG antibody M2 linked to 30 µl of protein A- or protein
G-Sepharose beads, respectively, for 3 h at 4 °C (Amersham
Biosciences). The beads were washed five times with Nonidet P-40 lysis
buffer and resuspended in denaturating sample buffer, and the eluted
IRF-3 proteins associated with CBP were analyzed by immunoblotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1, IFN
, and RANTES (Fig.
1B). Overexpression of wtIRF-3 alone minimally induced
IFN
1, IFN
, and RANTES promoter activities, as demonstrated previously (10, 21, 31), whereas introduction of the C-terminal point
mutation, S396D, enhanced IRF-3 transactivating potential over wtIRF-3
by 13-, 14-, and 11-fold for the IFN
1, IFN
, and RANTES promoters,
respectively (Fig. 1B). The IFN
1, IFN
, and RANTES
promoters were also activated by the double point mutant 2D (13-, 12-, and 12-fold over wtIRF-3, respectively) and, as previously reported
(10, 16, 18, 21), the multiple point mutant 5D (9-, 5.5-, and 8-fold
induction, respectively). However, the point mutants S398D,
S398D/S402D, and S402D/S404D/S405D exhibited only intermediate effects,
and the S385D/S386D mutant did not induce these promoters. Our initial
result thus demonstrates that substitution of Ser396 with
the phosphomimetic Asp is sufficient to generate a constitutively active form of IRF-3 that functions as a strong activator of promoters containing PRD I-III or ISRE regulatory elements.
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Fig. 1.
IRF-3 S396D phosphomimetic is a strong
transactivator of PRD I-III- and ISRE-containing promoters.
A, schematic representation of IRF-3 and location of the
point mutants used in this study. The DNA-binding domain, the
nuclear export sequence element, the proline-rich region, and
the C-terminal IRF association domain are indicated. The region between
amino acids 382 and 414 is expanded below the schematic. The amino
acids targeted for alanine or aspartic acid substitutions are shown in
large letters. The point mutations are indicated below the
sequence: S396D; S396A; S398D; S398A; 2D, S396D and S398D; 2A, S396A
and S398A; S398D/S402D; S398A/S402A; 3D, S402D, T404D, and S405D; 3A,
S402A, T404A, and S405A; 5D, S396D, S398D, S402D, T404D, and S405D; 5A,
S396A, S398A, S402A, T404A, and S405A; J2D, S385D and S386D; and J2A,
S385A and S386A. B, HEK 293 cells were transiently
transfected with luciferase reporter constructs containing the IFN-
promoter (left panel), the IFN-
enhancer (middle
panel), and the RANTES promoter (right panel) and
different IRF-3 point mutants as indicated below the bar graphs. At
36 h post-transfection, the luciferase activity was measured. The
relative luciferase activity was measured as fold activation over the
transfection of pFLAG-CMV2 alone. Each value represents the mean ± S.E. of triplicate determinations. The data are representative of at
least four different experiments with similar results.
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Fig. 2.
Ser396 is the minimal
phosphoacceptor site required for IRF-3 association with CBP
coactivator. HEK 293 cells were transiently transfected with
different FLAG-tagged IRF-3 constructs as indicated and 24 h
post-transfection were left untreated or infected with SeV (40 HAU/106 cells) for 12 h. WCE (500 µg) were
immunoprecipitated (IP) with anti-CBP antibody A22 absorbed
to protein A-Sepharose beads. The immunoprecipitated proteins were
resolved by SDS gel electrophoresis on 7.5% acrylamide gel and
transferred to nitrocellulose membrane. The membranes were probed with
anti-FLAG antibody. WCE (25 µg; 5% input) run on a 7.5% acrylamide
gel, transferred to membrane, and immunoblotted with anti-FLAG antibody
are also shown for both set of mutants: alanine (A) and
aspartic acid substitutions (B).
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Fig. 3.
Expression of IRF-3 S396D induces the
endogenous gene encoding ISG56. HEC 1B cells were transiently
transfected with the indicated FLAG-tagged IRF-3 phosphomimetic point
mutants. In one condition, HEC 1B cells were also infected for 15 h with SeV (40 HAU/106 cells). At 48 h
post-transfection, WCE (30 µg) were resolved by SDS gel
electrophoresis on 10% acrylamide gel and transferred to
nitrocellulose membrane. The membrane was probed with anti-ISG56
antibody (upper panel), stripped and reprobed with
anti-actin (bottom panel), and stripped and finally reprobed
with an anti-FLAG antibody (middle panel). Fold induction is
expressed as a ratio between the densitometric values for ISG56 and
actin expression level. The values were then normalized to
pFLAG-CMV2.
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[in a new window]
Fig. 4.
In vivo phosphorylation of
Ser396 of IRF-3 following virus infection.
A, HEK 293 cells were transiently transfected with
FLAG-tagged constructs as indicated. At 24 h post-transfection,
the cells were left untreated or infected with SeV (40 HAU/106 cells) for 7h. WCE (500 µg) were
immunoprecipitated with anti-FLAG antibody linked to protein
G-Sepharose beads. The immunoprecipitated proteins were resolved by SDS
gel electrophoresis on a 7.5% acrylamide gel and transferred to
nitrocellulose membrane. The membranes were probed with HIS5033
antibody. Following stripping, the membrane was reprobed with an
antibody that reacts against whole IRF-3 (Santa Cruz; SC-9082).
B, endogenous phosphorylation of IRF-3 on
Ser396. WCE (50 µg) prepared from HEK 293 cells
uninfected ( ) or infected with SeV (40 HAU/106 cells) for
the indicated times were resolved by 10% SDS-PAGE and transferred to
nitrocellulose. IRF-3 was analyzed by immunoblotting (IB)
for the presence of Ser396 phosphorylation with HIS5033
antibody (lanes 1-3). The membranes were stripped and
reprobed with the anti-IRF-3 antibody SC-9082 (lanes 4-6).
C, HEK 293 cells were transiently transfected with
expression plasmids encoding either SeV or measles virus N. At 36 h post-transfection WCE (50 µg) were analyzed as detailed above using
IRF-3 396 phosphospecific HIS5033 (upper panel) and
anti-IRF-3 antibody SC-9082 (middle panel). Expression of
SeV and measles virus N was analyzed by reverse transcriptase-PCR using
the full-length primers described under "Materials and
Methods."
B
by the I
B kinase complex was measured
using either the Ser32 phosphospecific antibody (Fig.
5F, lanes 2-8) or change of mobility in SDS gel
observed by the use of the I
B
-specific MAD 10 antibody (Fig.
5F, lanes 11-16). Together, these data
demonstrate that Ser396 phosphorylation of IRF-3 occurs
in vivo following treatment with inducers such as SeV and
poly(I-C), but not LPS.
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Fig. 5.
IRF-3 Ser396 is targeted by virus
and dsRNA in U373/CD14. WCE (45 µg) prepared from the U373
astrocytoma cell line that overexpressed the TLR4 coreceptor CD14
(U373/CD14) untreated ( ) or treated with SeV (40 HAU/106
cells) (A and D), poly(I-C) (100 µg/ml)
(B and E), and LPS (10 µg/ml) (C and
F) for the indicated time points were resolved on 7.5%
acrylamide gels and transferred to nitrocellulose membranes.
Ser396 phosphorylation of IRF-3 was detected with HIS5033
antibody in immunoblot analysis (A-C, lanes
1-8) and with anti-IRF-3 antibody SC-9082 (A-C,
lanes 9-16). The membranes were stripped and reprobed with
a phosphospecific antibody against I
B
(I
B
-P)
(D-F, lanes 1-8) and an antibody that
recognized nonphosphorylated and phosphorylated I
B
(MAD 10)
(D-F, lanes 9-16). The same results were
observed in three separate experiments.
B
(Fig. 6, C and D).
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Fig. 6.
IRF-3 Ser396 phosphorylation is
not detected in LPS treated U937 cells. WCE (45 µg) prepared
from the U937 cell line untreated ( ) or treated with SeV (40 HAU/106 cells) (A and C) and LPS (10 µg/ml) (B and D) for the indicated times were
resolved on SDS gels. The phosphorylation of IRF-3 and I
B
in U937
cells was measured as described in Fig. 5. These data are
representative of at least three experiments with similar
observations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank members of the Molecular Oncology Group at the Lady Davis Institute, McGill University for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by research grants from the Canadian Institutes of Health Research, the National Cancer Institute of Canada, and the Canadian Network for Vaccines and Immunotherapeutics.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ These authors contributed equally to this work.
Supported by a Canadian Institutes of Health Research
post-doctoral fellowship.
** Supported by a post-doctoral Fonds de la Recherche en Santé du Quebec (FRSQ) fellowship.
Supported by an National Science and Engineering Research
Council studentship.
¶¶ Supported by a FRSQ Chercheur Boursier.
Supporte by a Canadian Institutes of Health Research
Senior Scientist award. To whom correspondence should be addressed:
Lady Davis Institute-Jewish General Hospital, McGill University, 3755 Cote Ste. Catherine, Montreal, PQ H3T 1E2, Canada. Tel.:
514-340-8222 (ext. 5265); Fax: 514-340-7576.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M209851200
2 D. Duguay and J. Hiscott, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: IRF, interferon regulatory factor; IFN, interferon; LPS, lipopolysaccharide; STAT, signal transducers and activators of transcription; ISRE, interferon-stimulated response element(s); ds, double-stranded; CBP, cAMP-response element-binding protein-binding protein; PRD, positive regulatory domain(s); RANTES, regulated on activation normal T cell expressed and secreted; ISG, IFN-stimulated gene; N, nucleocapsid; SeV, Sendai virus; HEK, human embryonic kidney; TLR, Toll-like receptor; WCE, whole cell extracts; HAU, hemagglutination units.
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REFERENCES |
---|
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---|
1. | Akira, S., Takeda, K., and Kaisho, T. (2001) Nat. Immunol. 2, 675-680[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Samuel, C. E.
(2001)
Clin. Microbiol. Rev.
14,
778-809 |
3. | Servant, M. J., Grandvaux, N., and Hiscott, J. (2002) Biochem. Pharmacol. 64, 985-992[CrossRef][Medline] [Order article via Infotrieve] |
4. | Sen, G. C. (2001) Annu. Rev. Microbiol. 55, 255-281[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Sing, A.,
Merlin, T.,
Knopf, H.-P.,
Nielsen, P. J.,
Loppnow, H.,
Galanos, C.,
and Freudenberg, M. A.
(2000)
Infect. Immunity
68,
1600-1607 |
6. | Toshchakov, V., Jones, B. W., Prerera, P. Y., Thomas, K. W., Cody, M. J., Zhang, S., Williams, B. R. G., Major, J., Hamilton, T. A., Fenton, M. J., and Vogel, S. N. (2002) Nat. Immunology 3, 392-398[CrossRef][Medline] [Order article via Infotrieve] |
7. | Mamane, Y., Heylbroeck, C., Genin, P., Algarte, M., Servant, M. J., LePage, C., DeLuca, C., Kwon, H., Lin, R., and Hiscott, J. (1999) Gene (Amst.) 237, 1-14[CrossRef][Medline] [Order article via Infotrieve] |
8. | Servant, M. J., ten Oever, B., and Lin, R. (2002) J. Interferon Cytokine Res. 22, 49-58[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Yoneyama, M.,
Suhara, W.,
Fukuhara, Y.,
Fukada, M.,
Nishida, E.,
and Fujita, T.
(1998)
EMBO J.
17,
1087-1095 |
10. |
Lin, R.,
Heylbroeck, C.,
Pitha, P. M.,
and Hiscott, J.
(1998)
Mol. Cell. Biol.
18,
2986-2996 |
11. | Yoneyama, M., Suhara, W., Fukuhara, Y., and Fujita, T. (1997) J. Interferon Cytokine Res. 17, S53 |
12. |
Weaver, B. K.,
Kumar, K. P.,
and Reich, N. C.
(1998)
Mol. Cell. Biol.
18,
1359-1368 |
13. |
Servant, M. J.,
ten Oever, B.,
LePage, C.,
Conti, L.,
Gessani, S.,
Julkunen, I.,
Lin, R.,
and Hiscott, J.
(2001)
J. Biol. Chem.
276,
355-363 |
14. |
Smith, E. J.,
Marie, I.,
Prakash, A.,
Garcia-Sastre, A.,
and Levy, D. E.
(2001)
J. Biol. Chem.
276,
8951-8957 |
15. | Wathelet, M. G., Lin, C. H., Parekh, B. S., Ronco, L. V., Howley, P. M., and Maniatis, T. (1998) Mol. Cell 1, 507-518[Medline] [Order article via Infotrieve] |
16. |
Lin, R.,
Mamane, Y.,
and Hiscott, J.
(1999)
Mol. Cell. Biol.
19,
2465-2474 |
17. |
Kumar, K. P.,
McBride, K. M.,
Weaver, B. K.,
Dingwall, C.,
and Reich, N. C.
(2000)
Mol. Cell. Biol.
20,
4159-4168 |
18. |
Lin, R.,
Heylbroeck, C.,
Genin, P.,
Pitha, P. M.,
and Hiscott, J.
(1999)
Mol. Cell. Biol.
19,
959-966 |
19. |
Azimi, N.,
Tagaya, Y.,
Mariner, J.,
and Waldmann, T. A.
(2000)
J. Virol.
74,
7338-7348 |
20. |
Grandvaux, N.,
Servant, M. J.,
tenOever, B. R.,
Sen, G. C.,
Balachandran, S.,
Barber, G. N.,
Lin, R.,
and Hiscott, J.
(2002)
J. Virol.
76,
5532-5539 |
21. |
Lin, R.,
Genin, P.,
Mamane, Y.,
and Hiscott, J.
(2000)
Mol. Cell. Biol.
20,
6342-6353 |
22. |
Schafer, S. L.,
Lin, R.,
Moore, P. A.,
Hiscott, J.,
and Pitha, P. M.
(1998)
J. Biol. Chem.
273,
2714-2720 |
23. |
Ronco, L. V.,
Karpova, A. Y.,
Vidal, M.,
and Howley, P. M.
(1998)
Genes Dev.
12,
2061-2072 |
24. |
Iwamura, T.,
Yoneyama, M.,
Yamaguchi, K.,
Suhara, W.,
Mori, W.,
Shiota, K.,
Okabe, Y.,
Namiki, H.,
and Fujita, T.
(2001)
Genes Cells
6,
375-388 |
25. |
Navarro, L.,
and David, M.
(1999)
J. Biol. Chem.
274,
35535-35538 |
26. |
Kawai, T.,
Takeuchi, O.,
Fujita, T.,
Inoue, J.,
Muhlradt, P. F.,
Sato, S.,
Hoshino, K.,
and Akira, S.
(2001)
J. Immunol.
167,
5887-5894 |
27. |
Malakhova, O. A.,
Malakhov, M. P.,
Hetherington, C. J.,
and Zhang, D. E.
(2002)
J. Biol. Chem.
277,
14703-14711 |
28. | Shinobu, N., Iwamura, T., Yoneyama, M., Yamaguchi, K., Suhara, W., Fukuhara, Y., Amano, F., and Fujita, T. (2002) FEBS Lett. 517, 251-256[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Karpova, A. Y.,
Trost, M.,
Murray, J. M.,
Cantley, L. C.,
and Howley, P. M.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
2818-2823 |
30. |
tenOever, B. R.,
Servant, M. J.,
Grandvaux, N.,
Lin, R.,
and Hiscott, J.
(2002)
J. Virol.
76,
3659-3669 |
31. |
Juang, Y. T.,
Lowther, W.,
Kellum, M.,
Au, W. C.,
Lin, R.,
Hiscott, J.,
and Pitha, P. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9837-9842 |
32. |
Suhara, W.,
Yoneyama, M.,
Kitabayashi, I.,
and Fujita, T.
(2002)
J. Biol. Chem.
277,
22304-22313 |
33. | Guo, J., Peters, K. L., and Sen, G. C. (2000) Virology 267, 209-219[CrossRef][Medline] [Order article via Infotrieve] |
34. | Orr, S. L., and Tobias, P. (2000) J. Endotoxin Res. 6, 215-222[Medline] [Order article via Infotrieve] |
35. | Doyle, S., Vaidya, S., O'Connell, R., Dadgostar, H., Dempsey, P., Wu, T., Rao, G., Sun, R., Haberland, M., Modlin, R., and Cheng, G. (2002) Immunity 17, 251-263[Medline] [Order article via Infotrieve] |
36. | Fitzgerald, K. A., Palsson-McDermott, E. M., Bowie, A. G., Jefferies, C. A., Mansell, A. S., Brady, G., Brint, E., Dunne, A., Gray, P., Harte, M. T., McMurray, D., Smith, D. E., Sims, J. E., Bird, T. A., and O'Neill, L. A. (2001) Nature 413, 78-83[CrossRef][Medline] [Order article via Infotrieve] |
37. | Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., Takeuchi, O., Kobayashi, M., Fujita, T., Takeda, K., and Akira, S. (2002) Nature 420, 324-329[CrossRef][Medline] [Order article via Infotrieve] |
38. | O'Neill, L. A. (2002) Mol. Cell 10, 969-971[Medline] [Order article via Infotrieve] |
39. |
Weaver, B. K.,
Ando, O.,
Kumar, K. P.,
and Reich, N. C.
(2001)
FASEB J.
15,
501-515 |
40. | Sato, M., Suemori, H., Hata, N., Asagiri, M., Ogasawara, K., Nakao, K., Nakaya, T., Katsuki, M., Noguchi, S., Tanaka, N., and Taniguchi, T. (2000) Immunity 13, 539-548[Medline] [Order article via Infotrieve] |