(Received for publication, September 17, 1996, and in revised form, January 28, 1997)
From the Department of Food Engineering and
Biotechnology, Technion, Haifa 32000, Israel, the ¶ Laboratory
of Molecular Growth Regulation, NICHHD, National Institutes of Health,
Bethesda, Maryland 20892, the
Division of Cytokine Biology,
Center for Biologics Evaluation, Food and Drug Administration,
Bethesda, Maryland 20892-4555, and the ** Genetics of Eukaryotes,
GBF-Gesellschaft fur Biotechnologische Forschung mbH,
Mascheroder Weg 1,
D-38124 Braunschweig, Federal Republic of Germany
Two families of transcription factors mediate
interferon (IFN) signaling. The first family, signal transducers and
activators of transcription (STATs), is activated within minutes of IFN
treatment. Specific phosphorylation events lead to their translocation
to the nucleus, formation of transcriptional complexes, and the
induction of the second family of transcription factors termed
interferon regulatory factors (IRFs). Interferon consensus sequence
binding protein (ICSBP) is a member of IRF family that is expressed
only in cells of the immune system and acts as a transcriptional
repressor. ICSBP binds DNA through the association with other
transcription factors such as IRF-1 or IRF-2. In this communication,
the domain that is involved in protein-protein interactions was mapped
to the carboxyl terminus of ICSBP. This domain is also important for
mediating ICSBP-repressing activity. In vitro studies
demonstrated that direct binding of ICSBP to DNA is prevented by
tyrosine (Tyr) phosphorylation. Yet, Tyr-phosphorylated ICSBP can bind
target DNA only through the association with IRF-2 and IRF-1. This type of phosphorylation is essential for the formation of heterocomplexes. Tyr-phosphorylated ICSBP and IRF-2 are detected in expressing cells
constitutively, and Tyr-phosphorylated IRF-1 is induced by IFN-.
These results strongly suggest that like the STATs, the IRFs are also
modulated by Tyr phosphorylation that affects their biological
activities.
The activities of interferons (IFNs)1
is mediated mainly via successive phosphorylation events of IFN
receptors through specific receptor-associated tyrosine kinases that
belong to the Jak family and the subsequent phosphorylation of
transcription factors that translocate from the cytoplasm to the
nucleus. The transcription factors belong to the signal transducers and
activators of transcription (STAT) family of proteins (for review see
Refs. 1-3). In the case of IFN-/
signaling, mainly STAT1
(p91), STAT1
(p84), and STAT2 (p113) are phosphorylated on specific
Tyr residues allowing their association and translocation to the
nucleus. This complex, ISGF3
, associates in the nucleus with another
polypeptide termed ISGF3
(p48) that belongs to a different family of
transcription factors termed interferon regulatory factors (IRFs). The
activated factor, ISGF3, binds interferon-stimulated response element
(ISRE) containing promoters and serves as a transcriptional activator. In the case of IFN-
signaling, similar events occur on the receptor, yet mainly STAT1
is recruited which upon phosphorylation
translocates to the nucleus where it binds to gamma activation sequence
(for review see Refs. 1, 2, 4). Both Tyr phosphorylation and serine/threonine (Ser/Thr) phosphorylation, mediated by the
mitogen-activated protein kinase, are crucial for IFN signaling. Tyr
phosphorylation is critical for the translocation and the binding to
the DNA, and Ser/Thr phosphorylation is crucial for maximal
transcriptional activation (5, 6). This signaling cascade is
down-regulated upon activation of a specific set of Tyr phosphatases
that inactivate the receptors, the kinases, and the transcription
factors (7-9).
The delayed response to IFN is mediated mainly by the IRF family of
proteins that at least in part are induced by STATs. The members of
this family of factors share significant homology in the first 115 amino acids (aa) that comprise the DNA binding domain (DBD) and are
reflected by the ability to bind a similar DNA motif termed ISRE or,
alternatively, interferon consensus sequence (ICS). Among this family
are IRF-1, IRF-2, IRF-3, IRF-5, ISGF3, ICSAT/Pip (also named LSIRF,
IRF-4), and ICSBP. Some of the factors are hematopoietic specific
(ICSBP and ICSAT/Pip), and the others are expressed in various tissues
and cell lines. The expression is either constitutive or induced upon
treatment with IFNs or upon viral infection. Each individual member of
this family exerts distinct biological effects
(10-17).2 For example, IRF-1 acts as a
transcriptional activator and as a tumor suppressor gene, and its
inactivation may be linked to human hematopoietic malignancies. It is
also capable of exerting anti-proliferative effects and is involved in
apoptosis of mitogen-activated T lymphocytes (14, 19-26). On the other
hand, IRF-2 binds to similar DNA motifs such as IRF-1 and yet acts as a
repressor of IFN-stimulated genes and can induce oncogenic
transformation (15, 23, 24, 27). Recently, it was demonstrated that
IRFs can associate either with other family members (28, 29) or with other transcription factors (11, 30).
We have been working on the characterization of the IFN consensus
sequence binding protein (ICSBP) that is expressed exclusively in cells
of the immune system such as monocytes, B-cells, and T-cells (12, 13,
31). The expression of this protein is enhanced following exposure of
the cells to IFN- and to a lesser degree following exposure to
IFN-
. ICSBP functions as a repressor on promoters containing either
ICS motif or positive regulatory domain I (PRDI), a DNA motif located
on the promoter of IFN-
(13, 32). The repression mediated by this
factor can be alleviated by exposing the cells to IFNs (13, 31, 33).
ICSBP has a modular structure that includes the DBD and the repression
domain (28, 29). ICSBP can associate with IRF-1, IRF-2 (28, 29), and
PU.1 that belongs to the Ets family of transcription factors (11).
In this communication, we characterize the association of ICSBP with IRF-1 and IRF-2. The association domain was mapped to the carboxyl terminus and shows homology to other IRFs. This homology suggests a general role for this domain in mediating protein-protein interaction in this family of transcription factors. Deletion in this domain also affects the repression activity of ICSBP. Phosphorylation on Tyr residues (Tyr(P)) is essential for this association, yet Tyr phosphorylation also modulates the ability of each individual factor to bind target DNA sequences. Thus, in analogy to the STATs, phosphorylation events can modulate interaction of IRFs with other factors and alter DNA binding activity.
HeLa cells were obtained from ATCC (Rockville, MD) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Human monocytic U937 cells that are overexpressing ICSBP and U937 cells that were transfected with an empty vector were cultured in RPMI 1640 supplemented with 10% fetal calf serum and 400 µg/ml G418 (34).
PlasmidsFor transient co-transfection assays, the reporter
gene chloramphenicol acetyltransferase (CAT) driven by the basal human -globin promoter, to which four repeats of PRDI motif were
connected, was used (13). To generate expression vectors corresponding to the coding region of ICSBP and the DBD of ICSBP, the full-length human ICSBP and the segment corresponding to the first 121 aa were both
cloned into the mammalian expression vector, pcDNAI/Neo (Invitrogen) under the control of the cytomegalovirus promoter (pCMVICSBP and pCMVICSBPDBD, respectively). The expression vectors containing carboxyl-terminal truncations of ICSBP, pCMVICSBP377, and
pCMVICSBP363 were generated by polymerase chain reaction (PCR) and
cloned into pcDNAI/Neo (see below for details).
For in vitro translation the plasmids containing ICSBP, IRF-1, and IRF-2 under the bacteriophage T7 promoter were as described previously (29).
In Vitro Transcription and TranslationThe assays were performed as described previously (29). Plasmids containing ICSBP, IRF-1, and IRF-2 under the T7 promoter were linearized downstream to the coding region with the appropriate restriction enzyme. 5 µg of linearized plasmids were transcribed in vitro by T7 RNA polymerase using a commercial kit (Stratagene). Proteins were translated in vitro using rabbit reticulocyte lysate (RRL) system (Promega) according to the manufacturer's instructions. To block protein phosphorylation during the translation reaction, genistein (25 µg/ml) and/or staurosporine (75 nM) were added. To dephosphorylate Tyr residues, the translated protein was incubated with 2 units of Yop phosphatase at 30 °C for 30-60 min (New England Biolabs). To monitor translation efficiency, small scale reactions containing [35S]methionine were performed each time, and the labeled protein was separated on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to autoradiography.
In Vitro Generation of ICSBP Truncation and Deletion MutantsPCR was employed to generate in vitro
carboxyl-terminal or amino-terminal truncations of ICSBP as well as
internal deletions. For carboxyl-terminal deletions, the 5 primer
contained an engineered restriction site and 17 base pairs
corresponding to the T7 RNA polymerase recognition site. The 3
primers, 20-31 base pairs long, contained DNA sequences corresponding
to various positions on ICSBP as indicated in the text (see Fig.
2A, scheme) and engineered restriction site. A
33-aa amino-terminal deletion was generated using a 5
primer
containing the T7 RNA polymerase recognition site followed by ATG and
20-base pair sequence from amino acid residue 33. To generate internal
deletion, two segments of ICSBP were amplified separately. Segments
corresponding to the first 180 aa (or less) were amplified as above.
The other segments corresponding to the carboxyl end of ICSBP were
amplified using various 5
primers corresponding to the desired
internal deletions (see Fig. 2B, scheme) and a 3
primer corresponding to the end of the coding region. Following PCR
reactions, the correct size fragments were purified from agarose gels,
and 50 ng of each fragment was digested with engineered restriction
enzymes, mixed, and subjected to ligation. The primers were designed
such that an in frame fusion will occur. Following ligation, 1 µl of
the reaction was PCR-amplified with primers corresponding to the
beginning and the end of the coding region of ICSBP as above. The
correct size DNA fragments were purified from agarose gels, and 1 µg
of each fragment was transcribed in vitro and translated
in vitro as above. For each set of experiments a small scale
translation reaction was performed in the presence of
[35S]methionine, the translated proteins were separated
on a 10% SDS-PAGE and subjected to autoradiography to ensure that the
correct sizes and the expected amount of proteins were achieved. Each set of experiments was repeated at least three times with at least two
independent batches of PCR fragments. In all of these reactions we have
used Pfu (Stratagene) as the source of high fidelity
polymerase.
Electrophoretic Mobility Shift Assay (EMSA)
Gel shift reactions were carried out as described previously (25). A typical reaction contained 1-5 µl of in vitro translated proteins that were incubated in binding buffer (10 mM HEPES, pH 8.0, 5 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.025% bromphenol blue, 0,005% xylene cyanole, 10% Ficoll, 3% glycerol, 1 µg of sonicated poly[d(IC)], and 1 µg of sheared salmon sperm DNA) with at least 50,000 cpm of labeled trimer of PRDI motif (AAGTGA)3 for 10 min on ice. In supershift reactions, 1-2 µl of antiserum was incubated for 1 h with extracts, and the labeled probe in the binding buffer was added in the last 10 min. The samples were loaded on a pre-run 7% polyacrylamide gel. The dried gels were exposed to x-ray films.
DNA Transfection and CAT AssaysHeLa cells were transfected by the DNA coprecipitation assay using the modified calcium phosphate-mediated transfection procedure (35) with 2-4 µg of plasmid(s) DNA, and pUC19 was added as carrier DNA up to 20 µg. CAT assays were performed and normalized for protein concentration and transfection efficiencies as described previously (29). All experiments were repeated at least three times.
Preparation of Partially Purified bICSBPICSBP was cloned under the T7 promoter in the bacterial expression vector pET-3d, and large scale expression of the recombinant protein was performed according to manufacturer protocols (Novagen). The cells were harvested, and the pellet was resuspended in TE buffer in the presence of protease inhibitors and subjected to intensive sonication at 4 °C. Following centrifugation (10,000 × g for 10 min), the pellet was taken in 7 M guanidine HCl and sonicated again as above. The supernatant was diluted 8 times in EMSA binding buffer in the presence of protease inhibitors mixture. The preparation was dialyzed for 16 h at 4 °C against the same buffer, and the insoluble proteins were removed by centrifugation. This crude preparation was loaded over Sepharose-coupled ICS oligomer column as described (36), and the enriched preparation (~75%) was used for the binding experiments.
In Vitro Modification of bICSBP1 µg of bICSBP was incubated in 10 µl of RRL at different temperatures to test the effect of modification on DNA binding activity. During the various incubation periods, phosphorylation inhibitors such as genistein or staurosporine were added as above. The effect of Yop phosphatase (2 units at 30 °C) or alkaline phosphatase (1 unit at 37 °C, Boehringer Mannheim) was tested upon addition of the enzymes for 30-60 min to bICSBP preparations that were first incubated at 37 °C at various time intervals in RRL.
Immunoprecipitation and Western Blot AnalysesU937 cells or
U937 cells that are overexpressing ICSBP (34) were either not treated
or treated with IFN- (100 units/ml) for 5 h. The cells
(108/sample) were harvested, and the pellet was washed once
with phosphate-buffered saline and suspended in 200 µl of lysis
buffer (150 mM NaCl, 25 mM HEPES, pH 7.3, 1%
Triton X-100, 1 mM vanadate, 0.5 mM
dithiothreitol, 0.1% leupeptin, 0.1% aprotinin, 10 mM
-glycerol phosphate). Following 10 min incubation on ice with
occasional vigorous shaking, SDS was added to a final concentration of
1%, and the sample was incubated at 95 °C for 5 min and diluted 10 times with lysis buffer. 10 µl of agarose beads coupled with
antiphosphotyrosine monoclonal antibodies (4G10, UBI) were added and
incubated for 16 h at 4 °C with gentle agitation. The agarose
beads were washed twice with 1 ml of lysis buffer and finally incubated
for 5 min at room temperature with 15 µl of 100 mM phenol
phosphate. Following an additional wash in lysis buffer, the pellet was
taken in protein sample buffer and boiled for 5 min, and samples were
separated over 10% SDS-PAGE. The resolved proteins were transferred to
Immobilon-P (Amersham Corp.) and reacted with the various antibodies
using enhanced chemiluminescence (Amersham Corp.). Blots were reprobed
with various conjugated antibodies after the previous antibodies were
removed according to the manufacturer's protocol.
Previously, we demonstrated that ICSBP can associate
with IRF-1 or IRF-2 both in vivo and in vitro
(28, 29, 37). Fig. 1A demonstrates the
ability of ICSBP to associate with IRF-2 in vitro using RRL.
In vitro translated IRF-2 can bind a labeled trimer of PRDI
as detected by EMSA, whereas no shifted band is detected for in
vitro translated ICSBP (Fig. 1A, lanes 1 and
2, respectively). When both translated factors are mixed, a
new band corresponding to the heterocomplex is obvious (Fig. 1A
lane 3). To test the effect of phosphorylation on the association
between ICSBP and IRF-2, phosphorylation inhibitors (genistein and
staurosporine) were included during the translation step. In the
presence of the inhibitors some decrease in the DNA binding activity of
IRF-2 is observed while the DNA binding activity of ICSBP is still
undetectable (Fig. 1A, lanes 4 and 5). However,
when the two factors that were translated in the presence of
phosphorylation inhibitors are mixed, the band corresponding to
IRF-2·ICSBP complex is barely detected (Fig. 1A lane 6).
Thus, inhibition of phosphorylation results in a sharp decrease in
heterocomplex formation that is more profound than the observed
decrease in the intensity of the IRF-2 band. This implies that
heterocomplex formation is sensitive to the phosphorylation state of at
least one of the interacting factors.
To determine whether the phosphorylation state of both factors is equally important, we have mixed in vitro translated IRF-2 with ICSBP that was translated in the presence of genistein and staurosporine. It is clear that heterocomplex formation under these conditions is comparable with the one achieved in the absence of inhibitors (compare lane 3 to lane 7 in Fig. 1A). Conversely, we have mixed IRF-2 translated in the presence of the inhibitors with ICSBP. In this case, a significant decrease in heterocomplex formation is observed (compare lane 8 to either lane 7 or 3 in Fig. 1A) similar to the one detected when both factors were blocked for phosphorylation (compare lane 8 to lane 6 in Fig. 1A). This result suggests that only phosphorylated IRF-2 can effectively interact with ICSBP.
To test if phosphorylation events on Tyr residues are important for such association, we have incorporated a specific recombinant tyrosine phosphatase, Yersinia phosphatase (Yop), in the assay. It is clear from Fig. 1B that the ability of IRF-2 to bind DNA is only slightly decreased in the presence of the phosphatase (Fig. 1B lanes 2 and 5, respectively), whereas the ability to associate with ICSBP is severely impaired (Fig. 1B lanes 3-6, respectively). Dephosphorylation of IRF-2 on Tyr(P) is the reason for the decreased association of the two factors since dephosphorylation of ICSBP had no effect on heterocomplex formation, whereas dephosphorylation of IRF-2 resulted in marked decrease (Fig. 1B lanes 7 and 8, respectively). Similar results were obtained with IRF-1 indicating that the two factors interact with ICSBP in a similar manner (data not shown).
The Carboxyl-terminal Domain of ICSBP Is Required for the Association with IRF-2To map the domain responsible for the ability of ICSBP to associate with IRFs, a series of deletions were made at the carboxyl terminus as well as one deletion at the amino terminus (for details see "Experimental Procedures"). Fig. 2A demonstrates that deletions from position 377 to the carboxyl-terminal end of ICSBP had no effect on its ability to associate with IRF-2 (Fig. 2A lanes 1-9). However, the additional deletion of 14 or more aa from residue 377 toward the amino terminus prevented heterocomplex formation (Fig. 2A, lanes 10-13). This suggests that the carboxyl end of the association domain resides around position 377.
When the first 33 aa of ICSBP, which reside in the DBD, (ICSBPdel33) were deleted, no heterocomplex was observed (Fig. 2A, lane 15). Since ICSBPdel33 does not form heterocomplex with IRF-2, it is likely that the formation of heterocomplexes that bind to the PRDI is dependent upon intact DBDs of both interacting partners. Therefore, to map the amino-terminal end of the association domain, internal deletions were made in ICSBP (Fig. 2B). Deletion of 20 aa from positions 180-200 did not affect the ability of ICSBP to generate a heterocomplex with IRF-2 (Fig. 2B, lanes 1-5). Larger internal deletions of ICSBP (38 and 57 aa) prevented the interactions with IRF-2 (Fig. 2B, lanes 6-10), suggesting an amino-terminal border around aa 200. A deletion from position 200 to position 126 did not affect the ability of ICSBP to interact with IRF-2 (data not shown) indicating that it is not necessary for the formation of heterocomplexes. Similar results were obtained when the various deletion factors were reacted with IRF-1 indicating that the same region of ICSBP is responsible for the association with multiple IRFs (data not shown). The association domain of ICSBP encompasses over 177 aa between residues 200 and 377.
A Truncated ICSBP Defective in Its Ability to Associate with IRFs Is Also Defective in Its Repression ActivityTransient
cotransfection assays were performed in HeLa cells to determine the
repression activity of two carboxyl-terminal ICSBP truncations
(positions 363 and 377). The repression activity was compared with that
of full-length ICSBP construct driven by the CMV promoter or to the
ICSBP-DBD (first 121 aa). Repression activity was measured as the
minimal amount of transfected ICSBP that resulted in a significant
reduction in the enzymatic activity of the reporter gene, CAT, driven
by a promoter containing four repeats of the PRDI motif (38). As
previously demonstrated (13), a significant decrease (7-fold) in CAT
activity was observed with full-length ICSBP (Fig. 3,
compare two left bars). Equimolar amounts of the ICSBP-DBD
construct resulted in only a 2.3-fold reduction in CAT activity (Fig. 3
3rd bar). ICSBP-377 construct, which still retains
association with IRF-2, generated a reduction in CAT activity similar
to full-length ICSBP (6-fold, Fig. 3 compare the 5th and 2nd bars, respectively). However, cotransfection of
ICSBP-363 mutant confers CAT activity similar to the ICSBP-DBD
construct (Fig. 3, 4th and 3rd, bars,
respectively). These results suggest that part of the domain of ICSBP
implicated in heterocomplex formation is also important for maximal
repression activity.
The Interaction of ICSBP with DNA Is Dependent Upon Its Phosphorylation State
ICSBP shares high sequence similarity with
IRFs at the DBD. The ability of other IRF family members to bind
ISRE/PRDI motifs was demonstrated mainly by EMSA. As shown in Fig. 1,
in vitro translated ICSBP does not interact with DNA. In
addition, direct binding of ICSBP to DNA was not detected in nuclear
extracts prepared from different cell lines (28, 29). The specific
interactions of ICSBP with ISRE/PRDI motifs were more readily
demonstrated only by Southwestern analyses in which ICSBP was expressed
in Escherichia coli (12, 13). This suggested that the
inability of in vitro translated ICSBP or mammalian
expressed ICSBP to bind DNA is due to post-translational modifications
that do not take place in bacteria. To test this assumption, ICSBP was
expressed in E. coli, and the ability of the recombinant
protein to bind DNA was tested by EMSA. 1-2 µg of partially purified
bacterially expressed ICSBP (bICSBP, for details see "Experimental
Procedures") was reacted with a 32P-labeled trimer of
PRDI and analyzed by EMSA. Fig. 4A shows that a discrete band is detected with bICSBP that is not competed with 50-fold excess of mutant oligomer corresponding to the ICS of the major
histocompatibility complex class I but is competed with the native ICS
oligomer (Fig. 4A, lanes 1, 2, and 3,
respectively). The same band is still observed when bICSBP was
incubated in the presence of preimmune serum; however, this band is
supershifted when antiserum directed against ICSBP was included in the
EMSA (Fig. 4A, lanes 4 and 5, respectively).
Extract prepared from E. coli cells harboring the empty
expression vector did not demonstrate any bands by EMSA (data not
shown). These results indicate that bICSBP interacts with the DNA in a
specific manner.
The observed differences in the ability of bICSBP to bind DNA in comparison to in vitro translated ICSBP implies that post-translational modification can affect its interaction with the DNA. To further address this question, we incubated bICSBP in RRL at 37 °C for 30 or 60 min, and its ability to bind to the PRDI motif was tested by EMSA. After 30 min of incubation the intensity of binding was markedly decreased, and after 60 min of incubation the binding was essentially undetectable when compared with the same sample that was incubated at 4 °C (Fig. 4B, lanes 3, 4, and 2, respectively). The binding was not lost due to protein degradation since the same amount of protein was detected in all the samples by Western blot analysis (data not shown). No significant change in the DNA binding activity of ICSBP was observed in the absence or presence of RRL at 4 °C (Fig. 4B, lanes 1 and 2, respectively). However, when the samples that were incubated in RRL for 0, 30, and 60 min (Fig. 4B, lanes 2, 3 and 4, respectively) were incubated for an additional 30 min at 37 °C with alkaline phosphatase, robust DNA binding was observed in all three samples (Fig. 4B, lanes 5-7). This binding of bICSBP was even more intense than the DNA binding observed for the control (Fig. 4B, compare lanes 5-7 with lane 2). These results imply that phosphorylation of bICSBP occurs during incubation in RRL. These phosphorylations are probably preventing bICSBP from binding to the DNA. Treatment with alkaline phosphatase either removes all or some of the phosphate groups thus enabling the dephosphorylated factor to bind DNA even better than untreated bICSBP implying that some phosphorylation events may occur in bacterial cells.
To further characterize these phosphorylation events that are taking place in RRL, phosphorylation inhibitors were added as follows: genistein, which primarily blocks tyrosine (Tyr) phosphorylation at a concentration of 25 µg/ml, and staurosporine, which primarily blocks serine/threonine (Ser/Thr) phosphorylation at concentration of 75 nM. Fig. 4C shows the effect of these inhibitors on the DNA binding capability of bICSBP. The DNA binding of bICSBP following 30 min of incubation at 37 °C in RRL without the inhibitors is shown in Fig. 4C, lane 1. Under this condition bICSBP lost at least 50% of its DNA binding activity as shown in Fig. 4B, lanes 3 and 2, respectively. It is clear from Fig. 4C that when bICSBP is incubated in RRL in the presence of genistein, which blocks tyrosine phosphorylation, its DNA binding activity is enhanced as in the case of treatment with alkaline phosphatase (Fig. 4C compare lanes 2 and 3 with lane 1). In the presence of staurosporine, which blocks mainly serine/threonine phosphorylation, the DNA binding pattern of bICSBP resembled that of the untreated control sample that was incubated just with RRL (Fig. 4C, lanes 4 and 1, respectively). When both inhibitors were included, DNA binding was even weaker than that of the untreated control sample (Fig. 4C, compare lanes 1 and 5). The results suggest that RRL can support both tyrosine and serine/threonine phosphorylation and that bICSBP phosphorylation on Tyr residues inhibits DNA binding.
Phosphorylation on Tyrosine Residues Prevents ICSBP from Binding to DNAWe next wanted to demonstrate that phosphorylation on Tyr
residues accounts for the inability of ICSBP to bind DNA. For that purpose, bICSBP was incubated in RRL as described above, and the phosphorylated protein was then reacted with recombinant
Yersinia phosphatase that acts only on phosphorylated
tyrosine residues (for details see "Experimental Procedures") (39).
Fig. 5 clearly shows that DNA binding activity of bICSBP
is almost abolished following incubation in RRL (lanes 1 and
2, respectively). However, bICSBP did not lose its binding
activity when incubated further (30 °C for 60 min) in the presence
of Yop phosphatase (lane 3). Similar results were also
obtained with in vitro translated ICSBP (data not shown);
however, the amounts of translated product needed were much larger than
those used to demonstrate heterocomplex with IRF-2 as in Fig. 1. Thus,
these results confirmed that in RRL bICSBP is phosphorylated on
tyrosine residues, and this modification blocks its ability to bind
DNA.
ICSBP, IRF-1, and IRF-2 Are Tyrosine-phosphorylated in Vivo
The data presented above suggest that phosphorylation of
ICSBP might have a role in modulating its DNA binding ability. Thus, we
tested whether the factor is phosphorylated in vivo. For
that purpose we have used the promonocytic cell line, U937, in which ICSBP was stably transfected and expressed at high levels (34, 37).
Control cells or cells overexpressing the factor were treated for
5 h with IFN-, and the cells were lysed in the presence of 1%
SDS (to avoid protein complexes) and subjected to immunoprecipitation with monoclonal antibodies directed against Tyr(P) (for details see
"Experimental Procedures"). The precipitated proteins were separated on 10% SDS-PAGE and, following Western blot transfer, reacted with anti-ICSBP antibodies. The data in Fig. 6
show that a specific band was detected in cells that overexpressed the
factor. Phosphorylated ICSBP was not detected in parental U937 cells in which the factor is expressed at only moderate level. The absence of
phosphorylated ICSBP may be due to the low sensitivity of the combined
assays, immunoprecipitation and Western blot analysis.
To determine if other members of the IRF family of proteins are also
subjected to phosphorylation on Tyr residues, the same blot was reacted
with antibodies directed against IRF-1 and IRF-2. It is apparent from
Fig. 6 that in response to IFN treatment, a specific phosphorylated
IRF-1 band was induced in both cell lines tested. This induction
correlates with the reported induction of IRF-1 following treatment
with IFN- (31, 40). When the same blot was also reacted with
antibodies directed against IRF-2, a band with the expected molecular
mass appeared and its intensity was not affected by IFN treatment in
both cell lines. As a control to our combined
immunoprecipitation-Western analysis, the membrane was also reacted
with anti-STAT 1
(p91) antibodies (41). It is evident that indeed
p91 phosphorylation was induced in both cell lines following treatment
with the cytokine as previously reported (6, 42). Our results suggest
that members of the IRF family of proteins are phosphorylated on Tyr
residues. ICSBP and IRF-2 are constitutively phosphorylated, although
some increase in the phosphorylation state of ICSBP was observed
following treatment with IFN-
. The induction of IRF-1 expression
following exposure to the cytokine was accompanied by phosphorylation
on Tyr residues.
IRFs are a family of transcription factors that, like the STATs, mediate IFN signaling. Unlike the STATs that are activated within minutes following the binding of the ligand to the receptor, IRFs represent a secondary wave of response to the IFN signal. While STATs are activated through specific phosphorylation events (1-3), the role of phosphorylation in the modulation of the biological activity of IRFs has not been elucidated yet. Previously, we have demonstrated that ICSBP can interact with other members of the IRF family such as IRF-1 and IRF-2. Here we demonstrate that this interaction is dependent upon the phosphorylation state of the factor interacting with ICSBP. A 177-aa long domain on ICSBP is essential for this association and a deletion in that region affects the repressor activity of ICSBP. It is also demonstrated that the ability of ICSBP to bind to DNA is blocked through tyrosine phosphorylation. Finally we show that these members of the IRF family are phosphorylated in vivo on Tyr residues.
The association domain of ICSBP with IRF-1 and IRF-2 is located near
the carboxyl terminus between residues 200 and 377. Interestingly, this
177-aa long segment shares significant homology with ISGF3 as first
noted by Veals et al. (16). Moreover, this homology spans
over a region that is necessary for the interaction with ISGF3
subunit (i.e. STAT 1 and STAT 2). This lead to our initial search for interacting partners with ICSBP. As shown in Fig.
7, this homology is also found in ICSAT/Pip, IRF-3, and
IRF-5 suggesting that this conserved motif is probably essential for
the association of IRFs with other factors. Based on the functionality
of this domain in both ISGF3
and ICSBP, we propose to define it as
RFs ssociation omain (IAD). This
is in agreement with the finding that ICSAT/Pip can associate with PU.1
resulting in a transcriptional activation complex on the enhancer of
the immunoglobulin light chain. Similarly, ICSBP can associate with
this factor, but the nature of this complex has not been reported (11).
Since IRF-3 also shares similarity at this domain, it was postulated
that it might exert its activity via the formation of complexes that are similar to those of ISGF3 (43).
Our transfection studies also demonstrate that truncation of the association domain of ICSBP results in a reduced repression activity. Using domain swap analysis, we demonstrated that ICSBP has a modular structure that is comprised of two modules, a DBD and a repression domain (29). Here we show that a third functional module, the association module, overlaps at least in part with that of the repression domain. It is not clear if heterodimerization is a prerequisite for the repression activity of ICSBP. However, testing the IAD of either ICSBP or IRF-2 in a dominant negative assay will enable us to answer this question.
We show that in vitro phosphorylation of ICSBP on Tyr
residues prevents its ability to bind DNA. All IRF members share
significant homology at the DBD and can readily bind DNA with the
exception of ICSBP and the murine homologue of ICSAT, Pip (11, 29). This implies that the non-binding factors (ICSBP and Pip) may have
unique Tyr residues that are not conserved in the other IRF members.
ICSBP has 4 Tyr residues in the first amino-terminal 115 aa at
positions 23, 48, 107, and 110. The Tyr at position 110 is conserved
among all known IRFs, whereas Tyr at position 107 is conserved only in
ICSAT, ISGF3, and IRF-5. Since ISGF3
can bind DNA it is not
probable that this residue affects binding. However, Tyr at position 23 is shared only with IRF-5 and ICSAT, whereas Tyr at position 48 is
shared with only ICSAT. Since no information is available with respect
to the DNA binding ability of IRF-5, it is most probable that either
both residues or just one of them can prevent the DNA binding of at
least ICSBP and ICSAT upon phosphorylation. This does not exclude the
possibility that phosphorylation of Tyr residues outside the DBD might
also be involved in blocking ICSBP from binding to DNA. In an attempt to identify these Tyr residues, site-directed mutagenesis and domain
swap analysis with IRF-2 are being performed.
The data presented in this communication show that IRF-1, IRF-2, and
ICSBP can be phosphorylated in vivo. In U937 cells that are
overexpressing ICSBP, the protein is constitutively phosphorylated on
Tyr residues. Phosphorylated ICSBP was not detected in parental U937
cells although the protein can be detected by Western blot analysis
(data not shown) (18, 34). It is probable that our detection technique
that combines immunoprecipitation with Western blot analysis is not
sensitive enough. IRF-1 expression is induced following treatment with
IFN- (data not shown) (13, 40, 41). It is demonstrated here that
this induction of IRF-1 is accompanied by Tyr phosphorylation of the
protein. IRF-2, on the other hand, is constitutively phosphorylated.
The pattern of Tyr phosphorylation of the three IRFs tested implies
that phosphorylation events might play an important role in the
transcriptional activity of these factors.
In conclusion, we have demonstrated in this work that ICSBP can
associate with IRF-1 or IRF-2 through a specific domain, IAD, which is
conserved among some of the IRF members. This suggests a novel model
mechanism for the association of IRFs with other transcription factors.
In addition, it is demonstrated that the association between ICSBP and
IRFs is dependent upon phosphorylation of the interacting factor.
Phosphorylation events can also modulate the ability of ICSBP to bind
DNA; ICSBP can bind DNA either through association with other factors
or directly when not phosphorylated on Tyr residues. In response to
specific inducers, such as interferon, a signaling cascade is initiated
that also results in the induction of IRFs. These factors (also
phosphorylated on Tyr residues) influence gene expression and also
associate with ICSBP. This interaction in turn might affect their
biological activity. A further delayed response to IFN stimulation
involves the induction of specific phosphatases (7-9) which, for
example, dephosphorylate STAT1. It is feasible that these
phosphatases also act on ICSBP and IRF-1 or IRF-2 leading to
dissociation of the heterocomplexes from the DNA. Dephosphorylated
IRF-1 and IRF-2 bind DNA less effectively, whereas dephosphorylated
ICSBP becomes an active repressor that binds directly to the DNA and
down-regulates IFN-stimulated gene expression. Thus, phosphorylation
events might be responsible for the modulation of IRFs activities
either by promoting interaction with other transcription factors or by
enhancing or preventing binding to target DNA sequences.
We thank Drs. P. Wade and S. Kass for critical reading of the manuscript, with special thanks to Dr. A. Wollfe for his help and encouragement.