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Mol Cell Biol, May 1998, p. 2986-2996, Vol. 18, No. 5
Terry Fox Molecular Oncology Group,
Received 7 January 1998/Returned for modification 9 February
1998/Accepted 12 February 1998
The interferon regulatory factors (IRF) consist of a growing family
of related transcription proteins first identified as regulators of the
alpha beta interferon (IFN- Interferons (IFNs) are a large
family of multifunctional secreted proteins involved in antiviral
defense, cell growth regulation, and immune activation (63).
Virus infection induces the transcription and synthesis of multiple IFN
genes (33, 52, 63); newly synthesized IFN interacts with
neighboring cells through cell surface receptors and the JAK-STAT
signalling pathway, resulting in the induction of over 30 new cellular
proteins that mediate the diverse functions of the IFNs (17, 35,
39, 58). Among the many virus- and IFN-inducible proteins are the
growing family of interferon regulatory factor (IRF) transcription
factors, the IRFs. IRF-1 and IRF-2 are the best-characterized members
of this family, originally identified by studies of the transcriptional regulation of the human beta IFN (IFN- The presence of IRF-like binding sites in the promoter region of the
IFN- Several other members of the IRF family have been identified. The ICSBP
gene is expressed exclusively in the cells of the immune system
(18, 64), and its expression can be enhanced by IFN- Another unique member of the human IRF family, IRF-3, was recently
characterized (2). The IRF-3 gene encodes a 55-kDa protein which is expressed constitutively in all tissues. Recombinant IRF-3
binds to the ISRE element of the IFN-induced gene ISG-15 and stimulates
this promoter in transient expression assays. In contrast to IRF-1,
which contains both DNA binding and transactivating domains,
IRF-3 In the present study, we examined the virus-induced, posttranslational
modulation of IRF-3 protein following Sendai virus infection. Our
studies demonstrate that phosphorylation represents an important
posttranslational modification of IRF-3, leading to
cytoplasm-to-nucleus translocation of phosphorylated IRF-3, stimulation
of DNA binding and transcriptional activity, association of IRF-3 with
the transcriptional coactivator CBP/p300, and, ultimately, proteasome-mediated degradation.
Plasmid constructions and mutagenesis.
The IRF-3 expression
plasmid was prepared by cloning the EcoRI-XhoI
fragment containing the IRF-3 cDNA from the pSKIRF-3 plasmid downstream
of the cytomegalovirus (CMV) promoter of the CMVBL vector.
CMVt-IRF-3 was constructed by cloning of IRF-3 cDNA
downstream of the doxycycline-responsive promoter CMVt at
the BamHI site of the neoCMVtBL vector
(49). cDNAs encoding IRF-3 carboxyl-terminal deletion
mutations were generated by 28 cycles of PCR amplification with Vent
DNA polymerase. DNA oligonucleotide primers were synthesized with an
Applied Biosystems DNA/RNA synthesizer. The amino-terminal primer was
synthesized with an EcoRI restriction enzyme site, and the
carboxyl-terminal primers were synthesized with XbaI
restriction enzyme sites at their ends. The PCR products were purified
by phenol-chloroform extraction and ethanol precipitation, digested with EcoRI and XbaI, and inserted into
EcoRI/XbaI sites of the CMVBL vector. The point
mutations of IRF-3 were generated by overlap PCR mutagenesis with Vent
DNA polymerase. Mutations were confirmed by sequencing. The N-terminal
deletion mutations ( Generation of IRF-3 cell lines.
Plasmid
CMVt-rtTA (49) was introduced into 293 cells by
a calcium phosphate-based method. Cells were selected beginning at
48 h after transfection for about 1 week in alpha modified Eagle
medium ( Cell culture and transfections.
All transfections for
chloramphenicol acetyltransferase (CAT) assaying were carried out with
human embryonic kidney 293 cells or NIH 3T3 cells grown in Western blot analysis of IRF-3 modification and degradation.
To characterize the posttranslational regulation of the IRF-3 protein,
stable or transiently transfected IRF-3-expressing cells were infected
with Sendai virus (80 hemagglutinating units [HAU]/ml) or were
treated with 5 ng of tumor necrosis factor alpha per ml, either with or
without the addition of 50 µg of cycloheximide per ml. In some
experiments, the cells were treated with either 100 µM calpain
inhibitor I (ICN), 40 µM MG132 proteasome inhibitor, or an equivalent
volume of their respective solvents (ethanol) as a control. The cells
were washed with phosphate-buffered saline (PBS) and lysed in 10 mM
Tris-Cl (pH 8.0)-200 mM NaCl-1 mM EDTA-1 mM dithiothreitol
(DTT)-0.5% Nonidet P-40 (NP-40)-0.5 mM phenylmethylsulfonyl fluoride
(PMSF)-5-µg/ml leupeptin, 5-µg/ml pepstatin-5-µg/ml aprotinin. Equivalent amounts of whole-cell extract (20 µg) were subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in
a 10% polyacrylamide gel. After electrophoresis, the proteins were
transferred to a Hybond transfer membrane (Amersham) in a buffer
containing 30 mM Tris, 200 mM glycine, and 20% methanol for 1 h.
The membrane was blocked by incubation in PBS containing 5% dried milk
for 1 h and then was probed with IRF-3 antibody in 5% milk-PBS
at a dilution of 1:3,000. These incubations were done at 4°C
overnight or at room temperature for 1 to 3 h. After four 10-min
washes with PBS, the membranes were reacted with a peroxidase-conjugated secondary goat anti-rabbit antibody (Amersham) at
a dilution of 1:2,500. The reaction was then visualized with the
enhanced chemiluminescence detection system (ECL) as recommended by the
manufacturer (Amersham Corp.).
Phosphatase treatment.
Twenty to sixty micrograms of
whole-cell extract was treated with 0.3 U of potato acidic phosphatase
(PPA; Sigma) in a final volume of 30 µl of
piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer (10 mM PIPES [pH 6.0], 0.5 mM PMSF, 5-µg/ml
aprotinin, 1-µg/ml leupeptin, 1-µg/ml pepstatin) or 5 U of calf
intestine alkaline phosphatase (CIP; Pharmacia) in 30 µl of CIP
buffer. The phosphatase inhibitor mix contained 10 mM NaF, 1.5 mM
Na2MoO4, 1 mM Subcellular localization of GFP-IRF-3 proteins.
To analyze
the subcellular localization of wild-type and mutated forms of IRF-3
proteins in uninfected and virus-infected cells, the GFP-IRF-3
expression plasmids (5 µg) were transiently transfected into COS-7
cells by the calcium phosphate coprecipitation method. For virus
infection, transfected cells were infected with Sendai virus (80 HAU/ml
for 2 h) at 24 h post transfection. GFP fluorescence in
living cells was analyzed with a Leica fluorescence microscope with a
×40 objective.
EMSA.
Nuclear extracts were prepared from 293 cells at
different times after infection with Sendai virus (80 HAU/ml). In some
experiments, extracts were prepared from cells transfected with
different IRF-3 expression plasmids, as indicated in individual
experiments. The cells were washed in buffer A (10 mM HEPES [pH 7.9],
1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF) and were
resuspended in buffer A containing 0.1% NP-40. The cells were then
chilled on ice for 10 min before centrifugation at 10,000 × g. The pellets were then resuspended in buffer B (20 mM
HEPES [pH 7.9], 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2,
0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 5-µg/ml leupeptin, 5-µg/ml
pepstatin, 0.5 mM spermidine, 0.15 mM spermine, 5-µg/ml aprotinin).
Samples were incubated on ice for 15 min before being centrifuged at
10,000 × g. Nuclear extract supernatants were diluted
with buffer C (20 mM HEPES [pH 7.9], 20% glycerol, 0.2 mM EDTA, 50 mM KCl, 0.5 mM DTT, 0.5 mM PMSF). Nuclear extracts were subjected to
electromobility shift assaying (EMSA) by using a
32P-labeled probe corresponding to the PRDIII region of the
IFN- Immunoprecipitation and Western analysis of CBP-associated
proteins.
Whole-cell extracts (300 µg) were prepared from either
transfected or untransfected cells and precleared with 5 µl of
preimmune rabbit serum and 20 µl of protein A-Sepharose beads
(Pharmacia) for 1 h at 4°C. The extract was incubated with 10 µl of anti-CBP antibody A-22 (Santa Cruz) or 2 µl of anti-myc
antibody 9E10 (21) and 30 µl of protein A-Sepharose beads
for 2 to 3 h at 4°C. Precipitates were washed five times with
lysis buffer, which was eluted by boiling the beads for 3 min in 1×
SDS sample buffer. Eluted proteins were separated by SDS-PAGE and
transferred to Hybond transfer membranes. The membranes were incubated
with anti-IRF-3 (1:3,000) or anti-myc antibody 9E10 (1:1,000).
Immunocomplexes were detected by using a chemiluminescence-based
system.
Virus-induced phosphorylation of IRF-3 protein.
IRF-3 is
expressed constitutively in various cells, and its expression is not
enhanced by viral infection or by IFN treatment. To investigate whether
the IRF-3 protein is regulated by posttranslational modification after
virus infection, 293 cells were transiently transfected with an IRF-3
expression plasmid and subsequently infected with Sendai virus 24 h later. In cells transfected with the CMVBL vector alone, endogenous
IRF-3 protein was easily detected with a polyclonal IRF-3 antibody, and
in cells transfected with the IRF-3 expression plasmid, IRF-3 protein
levels were significantly increased (Fig.
1, lanes 1 and 3). Interestingly, Sendai
virus infection resulted in two alterations in the expression of IRF-3: an overall decrease in the amount of IRF-3 in transfected and control
cells (Fig. 1, lanes 2 and 4), and the generation of a more slowly
migrating form of IRF-3 (Fig. 1; compare lanes 1 and 2). In all
experiments, the turnover of IRF-3 after virus infection was more
pronounced with the endogenous protein than with the transfected
proteins (Fig. 1, as well as other figures). Because the transfected
proteins were driven by the CMV promoter, ongoing synthesis of
transfected IRF-3 may partially obscure the turnover of IRF-3.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Virus-Dependent Phosphorylation of the IRF-3
Transcription Factor Regulates Nuclear Translocation, Transactivation
Potential, and Proteasome-Mediated Degradation
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/
) gene promoters, as well as the
interferon-stimulated response element (ISRE) of some IFN-stimulated
genes. IRF-3 was originally identified as a member of the IRF family
based on homology with other IRF family members and on binding to the
ISRE of the ISG15 promoter. IRF-3 is expressed constitutively in a
variety of tissues, and the relative levels of IRF-3 mRNA do not change
in virus-infected or IFN-treated cells. In the present study, we
demonstrate that following Sendai virus infection, IRF-3 is
posttranslationally modified by protein phosphorylation at multiple
serine and threonine residues, which are located in the carboxy
terminus of IRF-3. A combination of IRF-3 deletion and point mutations
localized the inducible phosphorylation sites to the region
-ISNSHPLSLTSDQ- between
amino acids 395 and 407; point mutation of residues Ser-396 and Ser-398
eliminated virus-induced phosphorylation of IRF-3 protein, although
residues Ser-402, Thr-404, and Ser-405 were also targets.
Phosphorylation results in the cytoplasm-to-nucleus translocation of
IRF-3, DNA binding, and increased transcriptional activation.
Substitution of the Ser-Thr sites with the phosphomimetic Asp generated
a constitutively active form of IRF-3 that functioned as a very strong
activator of promoters containing PRDI-PRDIII or ISRE regulatory
elements. Phosphorylation also appears to represent a signal for
virus-mediated degradation, since the virus-induced turnover of IRF-3
was prevented by mutation of the IRF-3 Ser-Thr cluster or by proteasome
inhibitors. Interestingly, virus infection resulted in the association
of IRF-3 with the CREB binding protein (CBP) coactivator, as detected by coimmunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins. Mutation of
residues Ser-396 and Ser-398 in IRF-3 abrogated its binding to CBP.
These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and
IFN-responsive genes.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) gene (22, 23, 30, 47). Their discovery preceded the recent expansion of this group of IFN-responsive proteins, which now include seven other members, i.e., IRF-3, IRF-4/Pip/ICSAT, IRF-5, IRF-6, IRF-7, ISGF3
/p48, and
ICSBP (48). Structurally, the Myb oncoproteins share
homology with the IRF family, although the relationship of this family to the IFN system is unclear (62). Recent evidence also
demonstrates the presence of a virally encoded analog of cellular IRFs,
i.e., vIRF in the genome of human herpes virus 8 (55).
and - genes implicated the IRFs as essential mediators of
the induction of IFN genes. The original results of Harada et al.
(30, 32) indicated that IFN gene induction was activated by
IRF-1, while the related IRF-2 factor suppressed IFN expression. However, the essential role of IRF-1 and IRF-2 in the regulation of
IFN- and - gene expression has become controversial with the
observation that mice containing a homozygous deletion of IRF-1 or
IRF-2 or fibroblasts derived from these mice induced IFN- and -
gene expression after virus infection to the same level as that for the
wild-type mice or cells (44). On the other hand, IRF-1 was
shown to have an important role in the antiviral effects of IFNs
(44, 54). IRF-1 binds to the interferon-stimulated response
element (ISRE) present in many IFN-inducible gene promoters and
activates expression of some of these genes (54). However, activation of ISG genes by IFN- and - was shown to be mediated generally by the multiprotein ISGF3 complex (31, 36, 38). The binding of this complex to DNA is mediated by another member of the
IRF family, ISGF3/p48, which in IFN-treated cells interacts with
phosphorylated STAT1 and STAT2 transcription factors, forming the
heterotrimeric complex ISGF3 (8, 39, 62). The homozygous deletion of p48 in mice abolished the sensitivity of these mice to the
antiviral effects of IFNs, and virus-infected macrophages from
p48
/ mice showed an impaired induction of IFN- and
- genes (31).
.
ICSBP was shown to form a complex with IRF-1 and to inhibit the
transactivating activity of IRF-1 (9, 59). The homozygous
deletion of ICSBP in mice leads to defects in myeloid cell lineage
development and chronic myelogenous leukemia (34). Another
lymphoid cell-specific IRF, Pip/LSIRF/IRF-4, that interacts with
phosphorylated PU.1, a member of the Ets family of transcription factors (15), was identified (19, 43, 66). The
Pip/PU.1 heterodimer can bind to the immunoglobulin light-chain
enhancer and function as a B-cell-specific transcriptional activator.
Expression of Pip/LSIRF was induced by antigenic stimulation but not by
IFN, and Pip/LSIRF/IRF-4/ mice failed to develop mature
T and B cells (46). A novel member of the IRF family was
recently identified by its ability to bind to an ISRE-like element in
the promoter region of the Qp gene of Epstein-Barr virus
(69).
like IRF-2 and ICSBPdoes not contain a well-defined transactivation domain. Viral induction or IFN treatment does not
stimulate expression of the IRF-3 gene. In previous studies, we showed
that IRF-3 binds to the IE and PRDIII regions of the IFN- and -
promoters, respectively, but has different effects on their
transcriptional activity (56). While the induction of the
IFN-4 promoter activated by IRF-1 or virus infection was inhibited
in the presence of IRF-3, the fusion protein containing the IRF-3 DNA
binding domain and the RelA(p65) transactivation domain effectively
activated both IFN- and - promoters. In contrast, coexpression of
IRF-3 and RelA plasmids transactivated the IFN- gene promoter, but
not the promoter of the IFN-4 gene (56).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
N, N2A, N3A, and N5A) of IRF-3 were
generated by digestion of the related IRF-3/CMVBL plasmid with
BamHI (filled in with Klenow enzyme), partial digestion with
ScaI, and religation. Green fluorescence protein
(GFP)-IRF-3 expression plasmids were generated by cloning of cDNAs
encoding wild-type or mutated forms of IRF-3 into the downstream region
of EGFP in the pEGFP-C1 vector (Clontech). For construction of plasmids
encoding myc-tagged CREB-binding protein (CBP)-truncated proteins, the
cDNAs coding for CBP were generated from the pRC-RSV/mCBP plasmid
(provided by Dimitris Thanos) by PCR amplification. The cDNA fragments
were cloned in the downstream of region myc tag in the 5' myc-PCDNA3
vector (provided by Stephane Richard).
MEM; GIBCO-BRL) containing 10% heat-inactivated calf serum,
glutamine, antibiotics, and 2.5 ng of puromycin (Sigma) per µl.
Resistant cells carrying the CMVt-rtTA plasmid (rtTA-293 cells) were then transfected with the CMVt-IRF-3 plasmid.
Cells were selected beginning at 48 h for a period of
approximately 2 weeks in MEM containing 10% heat-inactivated calf
serum, glutamine, antibiotics, 2.5 ng of puromycin per µl, and 400 µg of G418 (Life Technologies, Inc.) per ml.
MEM (293 cells) or Dulbecco's MEM (NIH 3T3 cells; GIBCO-BRL) supplemented with
10% calf serum, glutamine, and antibiotics. Subconfluent cells were
transfected with 5 µg of CsCl-purified CAT reporter and expression
plasmids by the calcium phosphate coprecipitation method (293 cells) or
Lipofectamine (NIH 3T3 cells). The reporter plasmids were the
SVo CAT and ISG15 CAT reporter genes (56);
the transfection procedures were also previously described (41,
56). For individual transfections, 100 µg (SVo
CAT) or 10 µg (ISG15 CAT) of total protein extract was assayed for 1 to 2 h at 37°C. CAT activities were normalized with the
-galactosidase (-Gal) assay. All transfections were performed
three to six times.
-glycerophosphate, 0.4 mM
Na3VO4, and 0.1 µg of okadaic acid per ml.
promoter (5'-GGAAAACTGAAAGGG-3') or the ISRE region
of the ISG-15 promoter (5'-GATCGGGAAAGGGAAACCGAAACTGAAGCC-3').
The resulting protein-DNA complexes were resolved by 5% PAGE and
exposed to X-ray film. To demonstrate the specificity of protein-DNA
complex formation, 125-fold molar excess of unlabeled oligonucleotide was added to the nuclear extract before adding labeled probe.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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FIG. 1.
Sendai virus infection induces IRF-3 degradation. IRF-3
expression plasmid CMVBL-IRF3 (lanes 1 and 2) or CMVBL vector alone
(lanes 3 and 4), both at 5 µg, was transiently transfected into 293 cells by the calcium phosphate method. At 24 h posttransfection,
the cells were infected with Sendai virus for 16 h (lanes 2 and 4)
or were left uninfected (lanes 1 and 3). Whole-cell extracts (20 µg)
were prepared and analyzed by immunoblotting with anti-IRF-3
antibody.
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FIG. 2.
Sendai virus-induced phosphorylation and degradation of
IRF-3 protein. (A) rtTA-IRF-3 cells, which were selected as described
in Materials and Methods, were induced to express IRF-3 by doxycycline
treatment for 24 h. At 24 h after doxycycline addition, the
cells were infected with Sendai virus for 4, 8, 12, 16, 20, or 24 h (lanes 2 to 7) or were left uninfected (lane 1). IRF-3 protein was
detected in whole-cell extracts (10 µg) by immunoblotting. Two forms
of IRF-3, which were designated form I and form II, were detected. (B)
At 24 h post-Dox induction, rtTA-IRF-3 cells were infected with
Sendai virus for 16 h (lanes 4 to 8) or were left uninfected
(lanes 1 to 3). Whole-cell extracts from untreated cells (20 µg) or
Sendai virus-infected cells (60 µg) were incubated with 0.3 units of
PPA (lanes 2, 3, 7, and 8) or 5 U of CIP (lanes 4 and 5) in the absence
(lanes 1, 2, 4, 6, and 7) or presence (lanes 3, 5, and 8) of
phosphatase inhibitors. Phosphorylated IRF-3 protein appears as a
distinct band in immunoblots, migrating more slowly than IRF-3 forms I
and II.
Mapping the IRF-3 phosphorylation sites.
A series of deletions
of IRF-3 were generated to identify the virus-induced phosphorylation
site(s) of IRF-3 (Fig. 3A). 293 cells
were transiently transfected with IRF-3 deletion mutants, and
virus-mediated phosphorylation was measured by immunoblotting (Fig.
3B). The results indicated that virus-induced phosphorylation of IRF-3
occurs at the C-terminal end of IRF-3, since the mutations that
contained only the N-terminal part of IRF-3 protein (133, 240, 328, 357, or 394 amino acids [aa]) were not phosphorylated (Fig. 3B and
data not shown). Full-length and 407-aa forms of IRF-3 were
phosphorylated as a consequence of virus infection (Fig. 3B, lanes 1 to
4). C-terminal truncation of IRF-3 to a protein of 394 or 357 aa
removed the site(s) of inducible phosphorylation (Fig. 3B, lanes 5 to
8), although the shortened versions of forms I and II were still
observed. Also, in the IRF-3 9-133 mutation (N) from which the
DNA binding, N-terminal amino acids (aa 9 to 133) were removed, both
virus-induced phosphorylation of IRF-3 and the differential migration
of the shortened forms I and II were easily detected (Fig. 3B, lanes 9 and 10). Degradation of the endogenous forms of IRF-3 by virus
infection was also detected in this experiment (Fig. 3B; compare lanes
7 and 9 with lanes 8 and 10). Thus, by deletion analysis, a
phosphorylation domain of IRF-3 protein was localized to the region
-ISNSHPLSLTSDQ- between aa 395 and 407. Point mutations in the several putative Ser and
Thr phosphorylation residues within this region were generated in the
full-length protein and the 9-133 (N) protein (Fig.
4A). In the IRF-3 cDNA encoding these
proteins, the Ser-396, Ser-398, Ser-402, Thr-404, and Ser-405 residues
were replaced by alanine (5A), as were the three residues Ser-402,
Thr-404, and Ser-405 (3A) and the two residues Ser-396 and Ser-398
(2A). Transfection of these plasmids into 293 cells and subsequent
virus infection revealed that full-length, wild-type IRF-3 was
phosphorylated (Fig. 4B, lanes 4 and 8), whereas the IRF-3 proteins
containing the 2A and 5A mutations were no longer phosphorylated in
virus-infected cells (Fig. 4B, lanes 6 and 10). Interestingly, IRF-3-3A
was also very weakly phosphorylated as a consequence of virus
infection, thus implicating Ser-402, Thr-404, and Ser-405 as potential
secondary sites of phosphorylation. With the N IRF-3 protein and the
relevant point mutations, phosphorylation was detected with N (Fig.
4B, lane 12) but not with N-2A and N-5A (Fig. 4B, lanes 14 and
18); likewise, N-3A displayed very weak phosphorylation (Fig. 4B, lane 16). These experiments thus implicate Ser-396 and Ser-398 as
critical sites of virus-induced phosphorylation of IRF-3; however, residues Ser-402, Thr-404, and Ser-405 also contribute to the observed
phosphorylation, since the migration of phosphorylated N-3A is
significantly faster than that of N and the phosphorylation level is
decreased (Fig. 4B, lanes 12 and 16). Another study suggested the
involvement of the Ser residues at aa 385 and 386 as potential phosphoacceptor sites (67); in studies with the S385A and
S386A mutation, we found no evidence for inducible phosphorylation at these sites (40a). However, since these sites represent
consensus sites for CKI and CKII, constitutive phosphorylation is a
possibility.
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IRF-3 phosphorylation induces cytoplasmic to nuclear translocation
of IRF-3.
Initial studies indicated that IRF-3 was localized in
the cytoplasm of uninfected cells (67); to investigate the
role of phosphorylation on IRF-3 localization, wild-type and
point-mutated forms of IRF-3 were linked to GFP, transfected into COS-7
cells, and examined for Sendai virus-induced changes in subcellular
localization (Fig. 5). In uninfected
cells, GFP-IRF-3 localized exclusively to the cytoplasm; Sendai virus
infection resulted in translocation of IRF-3 to the nucleus within
8 h in 90 to 95% of the cells (Fig. 5A and B). Mutation of the
Ser-Thr cluster in GFP-IRF-3(5A) completely abrogated virus-induced
cytoplasm-to-nucleus translocation (Fig. 5C and D). Interestingly, the
substitution of the Ser-Thr cluster with the phosphomimetic Asp in
GFP-IRF-3(5D) likewise altered subcellular localization. IRF-3(5D)
localized both to the nucleus and to the cytoplasm in uninfected cells
(Fig. 5E), while virus infection resulted in an intense nuclear pattern
of IRF-3(5D) fluorescence (Fig. 5F). Point mutation of a putative
nuclear export signal in IRF-3, the L145A-L146A modificationtermed
IRF-3(nuclear export signal [NES])also changed subcellular
localization of IRF-3. In uninfected cells, GFP-IRF-3(NES) was
localized to the nucleus and cytoplasm, with a homogeneous,
extranucleolar pattern of nuclear staining. After virus infection,
GFP-IRF-3(NES) localized to the nucleus with an intense speckled
pattern of nuclear fluorescence in >95% of the cells, suggesting that
IRF-3(NES) may be trapped in the nucleus associated with the nuclear
pore complex.
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Transactivation of PRDI-PRDIII and ISRE promoters by IRF-3.
Next, the capacity of IRF-3 to regulate gene expression was analyzed by
transient transfection in human 293 and murine NIH 3T3 cells by using
the IFN- and ISG-15 promoters in reporter gene assays. Expression of
NF-
B RelA(p65), IRF-1, and IRF-3 alone minimally induced IFN-
promoter activity by between three- and fourfold (Fig.
6A and B), as shown previously (24,
56, 61). Introduction of the C-terminal point mutantsIRF-3(2A),
IRF-3(3A), and IRF-3(5A)reduced the low transactivation capacity of
IRF-3 to control levels (Fig. 6A). Interestingly, deletion of the
C-terminal 20 aa of IRF-3 to IRF-3(407) stimulated IFN- activity by
about 6-fold, which was indicative of the removal of an inhibitory
domain in IRF-3. However, further deletion to 394, 357, or 240 abrogated transactivation potential (Fig. 6A). Mutation of the NES
element was not sufficient to stimulate IFN- activity. Strikingly,
the substitution of the Ser-Thr cluster at aa 396 to 405 in IRF-3 with
the phosphomimetic Asp generated a very strong, constitutive transactivator protein that alone stimulated the IFN- promoter by
90-fold.
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promoter
requires synergistic activation by NF-B and IRF proteins (24,
61). To analyze the properties of IRF-3 in synergistic activation
of the IFN- promoter, coexpression studies were performed with
RelA(p65) expression plasmid and different wild-type and mutant forms
of IRF-3 (Fig. 6B). Coexpression of RelA and IRF-1 or RelA and IRF-3
stimulated IFN--CAT activity by 20- to 25-fold. IRF-3(407) and
RelA(p65) stimulated IFN- activity by about 40-fold, supporting the
idea of the removal of an inhibitory domain in IRF-3, whereas both
IRF-3(394) and the IRF-3(NES) failed to synergize with RelA in the
activation of the IFN- promoter. RelA and IRF-3(NES) produced a
relatively weak 8-fold induction of IFN- expression, indicating that
nuclear localization is not sufficient for IRF-3 activation. The
combination of RelA and IRF-3(5D) produced an 80-fold stimulation of
IFN- promoter activity (Fig. 6B); together with the data above,
IRF-3(5D) alone appears to be capable of full stimulation of the
IFN- promoter and further synergy with RelA is not observed (Fig. 6;
compare panels A and B). Surprisingly, IRF-3(5A) and RelA produced a
30-fold stimulation, suggesting that 5A can still synergize with RelA,
despite mutation of the Ser-Thr cluster.
The transactivation potential of IRF-3 was also analyzed by using the
ISG-15 promoter, an ISRE-containing regulatory element (Fig. 6C). As
shown previously (2) and as described above for the IFN-
promoter, IRF-3 alone weakly activated the ISG-15 promoter; in
the context of this regulatory element, IRF-3 was weaker than IRF-1,
which produced a 9-fold stimulation. Again, deletion of the C-terminal
20 aa of IRF-3 generated a protein that stimulated gene expression;
with the ISG-15 promoter, a 12-fold induction was observed; IRF-3(357)
did not stimulate gene expression but rather slightly repressed
ISG-15. Again remarkably, IRF-3(5D) produced a 50-fold induction of
the ISG-15 promoter (Fig. 6C), thus demonstrating that substitution of
the Ser-Thr sites with the phosphomimetic Asp generated a
constitutively active form of IRF-3 that functioned as a very strong
activator of promoters containing PRDI-PRDIII or ISRE regulatory
elements.
Inhibition of IRF-3 degradation.
Another consequence of virus
infection is the degradation of the IRF-3. Since phosphorylation of
proteins is functionally associated with the process of protein
degradation via the ubiquitin-dependent proteasome pathway (53,
57, 60), the effect of proteasome inhibitors on virus-induced
turnover of IRF-3 was examined. In cells transfected with the N and
N5A forms of IRF-3, virus-induced degradation of full-length
(endogenous) forms of IRF-3 (Fig. 7A, lanes 1 and 4) and the truncated N (Fig. 7B, lanes 1 and 4) was detected. Addition of the protease inhibitor calpain inhibitor I or the
proteasome inhibitor MG132 blocked virus-induced IRF-3 degradation
(Fig. 7A and 7B, lanes 4 to 6). Particularly with the N protein,
accumulation of the phosphorylated form of N was also detected in
virus-infected cells (Fig. 7B, lanes 5 and 6), suggesting that
phosphorylation of IRF-3 may represent a signal for subsequent
degradation by the proteasome pathway. To confirm this idea, the 5A
point-mutated form of IRF-3 was analyzed; the IRF-3-N5A protein was
resistant to virus-induced degradation (Fig. 7C, lanes 1 and 4); no
further stabilization of IRF-3-N5A occurred with calpain inhibitor I
or MG132 addition, and no phosphorylated IRF-3 was detected (Fig. 7C,
lanes 4 to 6). These experiments demonstrate that virus-dependent
phosphorylation of the C-terminal end of IRF-3 represents a signal for
subsequent proteasome-mediated degradation.
|
Interaction between IRF-3 and CBP in virus-infected cells.
To
examine the possibility that IRF-3 associated with the coactivator
CBP/p300 (Fig. 8A) following Sendai virus
infection, CBP was immunoprecipitated from virus-infected cells with
anti-CBP antibody; an immunoblot for IRF-3 revealed that IRF-3 was
coprecipitated from virus-infected cells but not from uninfected cells
(Fig. 8B, lanes 2 and 3). This interaction was observed clearly in
cells cotransfected with the IRF-3 expression plasmid (Fig. 8B, lane 3)
but was not seen when the immunoprecipitation was performed with
preimmune serum (Fig. 8B, lane 7). The endogenous IRF-3 also coprecipitated from virus-infected cells (Fig. 8B, lane 1). However, mutation of the Ser-Thr residues identified as the virus-inducible phosphorylation sites abrogated the association of IRF-3 with CBP. In
particular, IRF-3(2A) and IRF-3(5A) were detected in whole-cell extract
immunoblot but not in the CBP immunoprecipitate (Fig. 8B; compare lanes
4 and 6 with lanes 11 and 13). With the IRF-3(3A) mutant, interaction
with CBP was still observed (Fig. 8B, lane 5). The high background in
all lanes represents secondary antibody reactivity with rabbit
immunoglobulin G from the immunoprecipitation. Immunoblot analysis of
the whole-cell extracts revealed that phosphorylated IRF-3, as well as
forms I and II, was present in virus-infected cells (Fig. 8B, lane 10),
and in cells transfected with 2A, 3A, and 5A the forms I and II but not
the phosphorylated form of IRF-3 were observed (Fig. 8B, lanes 11 to
13). CBP has several domains that bind transcription factors, which
were designated CBP1, CBP2, and CBP3 (Fig. 8A [for a review, see
reference 29). To determine which domain of CBP
interacts with IRF-3, the three specific subdomains were myc tagged at
the 5' end by subcloning into the pCDNA3 vector (Fig. 8A). 293 cells
were cotransfected with these myc-tagged CBP expression plasmids
together with the IRF-3 N (9-133) expression plasmid. At 24 h after transfection, the cells were infected with Sendai virus,
coimmunoprecipitated with anti-myc antibody 16 h later
(21) and then immunoblotted for IRF-3. Endogenous IRF-3 and
transfected IRF-3 N proteins coprecipitated with CBP-3 from virus-infected cells but not from uninfected cells (Fig. 8C, lane 6).
In cells cotransfected with CBP-1 and CBP-2, no endogenous or
transfected N IRF-3 was detected (Fig. 8C, lanes 1 to 4). Immunoblot
analysis of the whole-cell extracts revealed that all three myc-tagged
CBP proteins were efficiently expressed in uninfected and
virus-infected cells (Fig. 8D). These results demonstrate that IRF-3
binds to the C-terminal domain of CBP in virus-infected cells and that
interaction with CBP requires phosphorylation of IRF-3 at Ser-396 and
Ser-398 but not at Ser-402, Thr-404, and Ser-405.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Several novel aspects of posttranslational regulation of the IRF-3 transcription factor are highlighted in the present study. (i) Sendai virus-dependent phosphorylation of IRF-3 occurring in a cluster of Ser and Thr sites in the carboxyl-terminal end of the protein was detected. The residues implicated in this regulatory phosphorylation event are Ser-396, Ser-398, Ser-402, Thr-404, and Ser-405, particularly the Ser-396 and Ser-398 residues. (ii) Phosphorylation of the IRF-3 in the Ser-Thr cluster resulted in cytoplasm-to-nucleus translocation of IRF-3; nuclear translocation was blocked by mutation of the phosphorylated amino acids. (iii) Sendai virus infection induced the DNA binding and transactivation potential of IRF-3. Furthermore, IRF-3 containing the phosphomimetic Asp at the sites of C-terminal phosphorylation was an exceptionally strong transactivator of PRDI-PRDIII- and ISRE-containing promoters. (iv) Phosphorylation was also required for the association of IRF-3 with the CBP coactivator protein. (v) Sendai virus infection resulted in IRF-3 degradation; again, phosphorylation was required as a signal for inducer-mediated degradation, since mutation of the Ser-Thr cluster also blocked virus induced degradation.
Cytoplasm-to-nucleus translocation of IRF-3 as a consequence of virus infection was inhibited by mutation of the Ser-Thr cluster, indicating an important regulatory role for C-terminal phosphorylation in the activation of IRF-3. Also strikingly, the conversion of the phosphorylation sites to the phosphomimetic Asp altered the subcellular localization of IRF-3 in uninfected cells. A proportion of IRF-3(5D) was localized to the nuclei of uninfected cells, suggesting that some IRF-3 may shuttle to and from the nucleus constitutively; this observation is consistent with the identification of a nuclear export signal in IRF-3. Mutation of L144A and L145A in the NES element produced the most impressive alterations in subcellular localization. In uninfected cells, IRF-3 was partitioned in both the nucleus and the cytoplasm; virus infection changed the nuclear pattern of staining from extranucleolar homogeneous staining as observed for wild-type IRF-3 to an intense nuclear speckling. At this stage, the nature of the subnuclear changes in IRF-3 localization are not explained, although it is possible that IRF-3(NES) translocates efficiently into the nucleus but becomes trapped in the nuclear pore complex during the export process.
One of the striking results of the mutagenesis of the C-terminal domain
of IRF-3 was the generation of IRF-3(5D), an exceptionally strong
activator of IFN- and ISG-15 gene expression. The phosphomimetic form of IRF-3 alone was able to stimulate IFN- expression as strongly as virus infection, a level of stimulation not previously been
observed in coexpression experiments (24, 61). In previous experiments, we demonstrated that IRF-3 was able to bind the ISRE element of ISG-15, as well as the PRDIII-PRDI and IE regions of the
IFN- and IFN- promoters, respectively (2, 56). Virus induction results in the appearance of two new protein-DNA complexes; supershift experiments confirmed that both complexes contain IRF-3 (32a); it is not clear at this stage whether the upper
complex also contains other proteins, such as in the VIC (10,
25) and DRAF (16) complexes, or whether the lower
complex represents a breakdown product of IRF-3. Strikingly, the same
complexes appeared following cotransfection of IRF-3(5D) expression
plasmid in the absence of virus induction, indicating that IRF-3(5D)
represented a constitutive DNA binding form of IRF-3. Thus, in
uninfected cells, IRF-3(5D) localized in part to the nucleus (Fig. 5),
interacted with DNA constitutively, and was a strong activator of gene
expression (Fig. 6).
The recent crystal structure of the related IRF-1 protein bound to PRDI
provides evidence for a novel helix-turn-helix motif that latches onto
a GAAA core sequence via three of the five conserved tryptophan amino
acids of the DNA binding domain (20). By analogy with IRF-3,
two GAAANN sequences present in PRDIII of IFN- and another GAAANN
element present in the PRDI may serve as DNA contacts for multiple
IRF-3(5D) proteins with strong activating potential. Similarly, the
ISRE element of the ISG-15 promoter also contains several GAAANN
anchors for potential IRF binding. Given the range of promoters that
possess this hexameric sequence (48), it will be of interest
to determine the capacity of IRF-3(5D) to stimulate expression of
different cytokine and chemokine genes.
IRF-3 joins a growing list of cellular and viral proteins that
functionally interact with CBP/p300 proteins, highly homologous proteins originally identified through their interactions with adenovirus E1A and CREB proteins (1, 13). As a critical
determinant of its global transcriptional coactivator activity,
CBP/p300 possesses histone acetyltransferase activity (5,
50). Acetylation of histones is involved in the destabilization
and remodeling of nucleosomes, which is a crucial step in permitting
the accessibility of transcriptional factors to DNA templates. Several
studies have now demonstrated that CBP/p300 participates in the
transcriptional process by providing a scaffold for different classes
of transcriptional regulators on specific chromatin domains (12,
50). A growing body of biochemical and genetic evidence also
implicates CBP/p300 as a negative regulator of cell growth, based on
its interactions with adenovirus E1a, simian virus 40 large T antigen,
and the tumor suppressor p53 among others. With regard to p53-CBP/p300 complex formation, functional interaction between these two important growth regulatory proteins accounts for several of the known activities of p53 (3, 29, 40); interestingly, CBP/p300 was shown
recently to acetylate p53 and to stimulate its transactivation
potential (28). It will be of interest to determine whether
IRF-3 is similarly modified by CBP association. The functional
consequences of IRF-3 interaction with CBP/p300 remain to be
elucidated, although recent studies demonstrated that CBP/p300 also
functionally interacts with STAT1 (68) and STAT2
(7) and may contribute to IFN- and IFN- nuclear
signalling. Elegant studies published during review of the manuscript
demonstrated that synergistic activation of the IFN- promoter
requires recruitment of CBP/p300 to the enhanceosome, via a new
activating surface assembled from the activation domains of all of the
transcription factors in the enhanceosome (37, 45).
Alterations in any of the activation domains decreased both CBP
recruitment and transcriptional synergy. By analogy, recruitment of
CBP/p300 to DNA-bound IRF-3 is likely required for maximal
transcriptional activation. Association requires the interaction of the
C-terminal domain of IRF-3 and the C-terminal interaction domain of
CBP, a region previously shown to associate with the p53 tumor
suppressor, whereas STAT1 and STAT2 associate with different regions of
CBP (7, 68).
Virus-induced phosphorylation of IRF-3 also represents a signal for
proteasome-mediated degradation of IRF-3, since mutation of Ser-396 and
Ser-398 or the use of proteasome inhibitors prevented postinfection
degradation of IRF-3. Virus-induced degradation of IRF-3 is reminiscent
of the virus-induced turnover of another member of the IRF family,
IRF-2. In response to double-stranded RNA (dsRNA) or viral induction,
the 50-kDa IRF-2 protein is proteolytically processed into a smaller,
24- to 27-kDa protein (51) comprising the 160-aa DNA-binding
domain (DBD) of IRF-2, which was termed TH3 (14) or In4
(65). Although TH3 has been shown to bind DNA and to repress
transcription more efficiently than the full-length IRF-2 protein
(42), its physiological role is not clear. Since the
induction kinetics of TH3 are slower than that of IFN- in response
to dsRNA or viral infection (14), it has been suggested that
the IRF-2 cleavage product is a postinduction repressor of IFN- gene
expression (65).
Virus-induced phosphorylation of IRF-3 at the C-terminal Ser and Thr
residues and its subsequent degradation by a proteasome-dependent pathway are also similar to the well-studied phosphorylation and degradation of IB, which leads to activation of NF-B binding activity (reviewed in references 4 and
6). In unstimulated cells, NF-B heterodimers are
retained in the cytoplasm by inhibitory IB proteins. Upon
stimulation by many activating agents, including cytokines, viruses,
and dsRNA, IB is rapidly phosphorylated and degraded, resulting
in the release and nuclear translocation of NF-B. The amino terminus
of IB represents a signal response domain for activation of
NF-B, and substitution of alanine for either Ser-32 or Ser-36
completely abolished the signal-induced phosphorylation and degradation
of IB and blocked activation of NF-B. These mutations also
blocked in vitro ubiquitination of the IB protein. The amino
terminus of IB is necessary for signal-induced phosphorylation
and ubiquitination, but for degradation to occur, there is an absolute
requirement for the C-terminal PEST domain (reviewed in references
4 and 6).
Similarities and differences between the observed degradation of IRF-3
and the mechanism of IB degradation exist. The C-terminal phosphorylation of IRF-3 as a consequence of virus infection is required for its subsequent degradation based on the deletion and point
mutation analysis of the region
-ISNSHPLSLTSDQ- between aa
395 and 407. Minimally, phosphorylation of Ser-396 and Ser-398 are
required for subsequent degradation, although Ser-402, Ser-404, and
Ser-405 may represent secondary phosphorylation sites. Likewise, in the
case of IB, phosphorylation of Ser-32 and Ser-36 are required for
inducer-mediated degradation. Furthermore, the protease inhibitor
calpain inhibitor I and the more specific proteasome inhibitor MG132
block IRF-3 turnover. A major difference in the mechanisms of IB
and IRF-3 turnover lies in the nature of the inducing stimuli. Multiple
inducerscytokines such as tumor necrosis factor and interleukin 1, viruses, lipopolysaccharide oxidative stress, etc. (6)all
lead to the induction of IB phosphorylation and degradation
whereas IRF-3 phosphorylation appears to be induced only by virus
infection and dsRNA addition; other inducers have not resulted in IRF-3
turnover (40a). A significant temporal difference also
exists between IB phosphorylation-turnover and IRF-3
phosphorylation-degradation. Many activators of NF-B stimulate
IB phosphorylation within minutes, and TNF-induced degradation
occurs within the first 15 to 30 min after treatment. In the case of
IRF-3, phosphorylation is not detected until 6 to 8 h after
infection and continues in a heterogeneous manner over the following 10 to 12 h. Previous experiments, however, have demonstrated that
Sendai virus-induced turnover of IB also occurs slowly over
several hours (24).
Based on the data presented in this study and by analogy with the
properties of other IRF family members (48), we propose the
following model to explain several observations. IRF-3 exists in a
latent state in the cytoplasm of uninfected cells; the C terminus may
physically interact with the DNA binding domain in such a way as to
obscure both the DBD and the IAD regions of the protein; the presence
of an autoinhibitory domain within the C-terminal 20 aa (407 to 427)
would explain the activating effect of this deletion, as seen
previously with IRF-4 (11, 19). Virus-induced phosphorylation at the Ser-Thr cluster at aa 396 to 405 leads to a
conformational change in IRF-3, exposing both the DBD and IAD and
relieving C-terminal autoinhibition. Translocation to the nucleus,
occurring via an unidentified nuclear localization sequence or in
conjunction with a transcriptional partner associating through the IAD
region, leads to DNA binding at ISRE- and PRDI-PRDIII-containing promoters. Phosphorylation is also necessary for IRF-3 association with
the chromatin-remodeling activity of CBP/p300. The presence of a NES
element ultimately shuttles IRF-3 from the nucleus and terminates the
initial activation of IFN-responsive promoters. The phosphorylated form
of IRF-3 exported from the nucleus may then be susceptible to
proteasome-mediated degradation. This scenario shares several features
with the protein synthesis-independent activation of NF-B and
further suggests that IRF-3 may represent a component of virus- or
dsRNA-inducible complexes such as DRAF (16) or VIC (10,
25) that could play a primary role in the induction of IFN or
IFN-responsive genes.
| |
ACKNOWLEDGMENTS |
|---|
We thank Dimitris Thanos, Stephane Richard, and Illka Julkunen for reagents used in this study. We also thank members of the Molecular Oncology Group, Lady Davis Institute, for helpful discussions.
This research was supported by grants from the Medical Research Council of Canada (J.H. and R.L.), the National Cancer Institute (J.H.), and the National Institutes of Health (P.M.P.). R.L. was supported in part by a Fraser Monat McPherson Fellowship from McGill University, C.H. was supported by a FRSQ/FCAR studentship, and J.H. was supported by an MRC Scientist award.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec, Canada H3T1E2. Phone: (514) 340-8222, 4509. Fax: (514) 340-7576. E-mail: mdli{at}musica.mcgill.ca.
| |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
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