(Received for publication, February 12, 1997, and in revised form, June 5, 1997)
From the Department of Pathology, University at Stony Brook, Stony Brook, New York 11794, and the § Division of Hematology, Mount Sinai School of Medicine, New York, New York 10029
Cells express a variety of STAT (signal
transducer and activator of transcription) transcription factors that
are structurally homologous and yet function specifically in response
to particular cytokines. The functions of the individual STATs are
dependent on distinct protein-protein interactions. STAT1 and STAT2 are activated by tyrosine phosphorylation in response to type I
interferons-/
(IFN-
/
) and subsequently form a multimeric
transcription factor designated the IFN-
-stimulated gene factor 3 (ISGF3). ISGF3 is a unique STAT complex because it also contains a
non-STAT molecule, p48, which is a critical DNA-binding component. We
provide evidence that STAT2 specifically interacts with p48 in
vivo before and after IFN-
stimulation. The specificity of
ISGF3 formation is therefore a result of the distinct nature of the
STAT2 molecule. Coimmunoprecipitation assays demonstrate p48
association with STAT2 but not STAT1. Hybrid STAT2·STAT1 molecules
were used to identify a region of STAT2 which specifically associates
with p48. The region of STAT2 interaction spans an amino-terminal
region of two predicted coiled coils. The studies demonstrate the
in vivo existence of a STAT2·p48 complex and a distinct
STAT2·STAT1 complex after IFN-
stimulation. Data suggest that
distinct bipartite complexes STAT2·p48 and STAT2·STAT1 translocate
to the nucleus and associate on the DNA target site as ISGF3.
Interferons (IFNs)1 play
a fundamental role in immune defense, they confer resistance to viral
infections, inhibit cellular proliferation, and activate a variety of
cells in the immune system (1, 2). IFNs elicit these physiological
responses by binding to cell surface receptors and transducing a signal
to the nucleus that activates expression of a subset of genes. The
discovery of a regulated signal transduction pathway in the IFN system
has served as a paradigm for receptor to nucleus signal transmission by
a variety of cytokines (3-6). A class of transcription factors which
resides in a latent state in the cytoplasm of the cell becomes activated by tyrosine phosphorylation in response to specific stimulation. These transcription factors have been designated signal
transducers and activators of transcription (STATs) (3). Seven distinct
human STAT molecules have been identified that can be phosphorylated by
tyrosine kinases of the Janus kinase family (6). Following
phosphorylation, the STATs can associate via their SH2 domains and
phosphotyrosine domains to form homodimers or heterodimers (7). The
STAT dimers translocate to the nucleus and bind to specific DNA
sequences in the promoters of stimulated genes. The DNA target site of
the STATs was first identified for the IFN--activated STAT1 as a
palindrome of GAAA residues (TTTCNNNGAAA) (3-5). The STAT molecules can bind to this consensus target site as
homodimers or heterodimers. Since distinct gene expression is induced
by different cytokines, many studies have focused on the intricate
question of specificity of STAT action.
One of the STAT factors, STAT2, is distinct in that it does not appear
to recognize a DNA target site as a homomeric complex. STAT2 was first
identified as a subunit of the multimeric transcription factor,
IFN--stimulated gene factor 3 (ISGF3), which forms in response to
IFN-
/
stimulation (8). ISGF3 is composed of two STATs, STAT1 and
STAT2, in association with a non-STAT molecule, p48 (8-10). p48 is a
member of a different family of transcription factors designated the
IFN regulatory factor family and is a critical DNA binding component of
ISGF3 (10). ISGF3 does not bind to a palindromic target site but rather
to a consensus tandem repeat of GAAA residues
(GGAAANNGAAAC) known as the IFN-
stimulated response element (ISRE) (3-5). Since transcriptional specificity is
regulated by precise protein-protein interactions acting in conjunction
with a particular DNA target site, we examined the protein-protein
association of the STATs with p48. Although the STAT molecules share
structural similarity, the unique association with p48 has led to the
identification of a particular domain in STAT2 which specifies
formation of ISGF3.
Human HeLa S3, HT1080, and HT1080 mutants (U3A and U6A, gifts of G. R. Stark, Cleveland Clinic Foundation Research Institute) were cultured in Dulbecco's modified Eagle's medium with 8% fetal bovine serum. Transfections were performed with calcium phosphate-DNA coprecipitates (11), and stable transformants were selected by resistance to G418 (250 µg/ml) and screened for STAT expression. Generation of hybridoma cell lines was performed by immunization of A/J mice with bacterially expressed p48 or STAT1 fusion proteins. Spleens from the mice were fused to NSO cells, and IgG1-producing hybridomas were selected (12-14).
Plasmid ConstructsReverse transcription-polymerase chain reaction of HeLa cell mRNA was performed with oligonucleotides that generated DNA corresponding to p48 (341-711nt) or STAT1 (1995-2378nt). These DNAs were cloned into an expression plasmid (pGEX, Pharmacia Biotech Inc.), and fusion proteins were derived for immunizations. STAT1 and STAT2 cDNAs (gift of J. E. Darnell, Jr., Rockefeller University) were cloned into the pcDNA3 eukaryotic expression vector (Invitrogen). STAT1/2 and STAT2/1 chimeras were generated by DNA cleavage and reciprocal ligation at an SphI site (corresponding to STAT1 325aa and STAT2 324aa). A T7 epitope was introduced into the STAT1/2 construct at position 825 by oligonucleotide insertion into the BalI site (15). The STAT1/2/1 chimera was constructed by polymerase chain reaction amplification (Pfu polymerase, Stratagene) of a STAT1 fragment and insertion into STAT2/1. STAT1/2/1 encodes STAT1 1-147aa, STAT2 148-324aa, and STAT1 325-750aa. Deletions of STAT2 were introduced by Bal-31 exonuclease digestion at a Bsu36I site (814nt/270aa). Resultant plasmid constructs were used to synthesize STAT2 fragments in vitro encoding 1-190aa (dl569-1128nt), 1-217aa (dl652-903nt), 1-230aa (dl689-887nt), or 1-250aa (dl749-886nt). The full-length p48 cDNA (gift of D. E. Levy, New York University) was cloned into the pGEX vector following polymerase chain reaction amplification to generate GST-p48 fusion protein.
Immunoprecipitation and Immunoblot AssaysCells were
treated with IFN- (500 units/ml) and IFN-
(100 units/ml) (gifts
of Hoffman-LaRoche) as described in the text. Protein extracts were
prepared after lysis in buffer containing 280 mM NaCl,
0.5% Nonidet P-40 (16). Approximately 4 mg of protein was incubated
with 4 µg of monoclonal anti-p48, anti-STAT1, anti-T7 (Novagen),
control MOPC141 (Sigma), or with 2 µg of polyclonal anti-STAT2 (Santa
Cruz Biotechnology) for 3 h at 4 °C. Immunocomplexes were
collected on protein G-agarose beads, separated by SDS-PAGE, and
electroblotted to Immobilon-P membrane (Millipore). Proteins were
detected after incubation with specific antibodies described above or
anti-phosphotyrosine (4G10, Upstate Biotechnology, Inc.) or anti-STAT2N
(Transduction Laboratories) and enhanced chemiluminescence reagents
(DuPont).
Bacterially expressed GST or GST-p48 protein was purified by binding to glutathione-agarose beads and eluted with reduced glutathione (Sigma) followed by dialysis in binding buffer (20 mM Hepes, pH 7.9, 100 mM KCl, 0.2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 0.0025% Nonidet P-40) (17). The STAT proteins were synthesized with a coupled transcription/translation system in the presence of [35S]methionine (TNT, Promega). Translation products were incubated with 7 µg of GST or GST-p48 protein immobilized on glutathione-agarose beads in binding buffer containing 1% milk and 0.1% Triton X-100 for 1-2 h at 4 °C. Bound proteins were eluted with SDS-gel loading buffer, separated by SDS-PAGE, and visualized by Enhance treatment (DuPont) and autoradiography.
Electrophoretic Mobility Shift AssayNuclear extracts were
prepared as described (18). DNA binding was performed with a
double-stranded oligonucleotide probe representing the ISRE site from
the ISG15 gene 5-GGGAAAGGGAAACCGAAACTGAA (
114/
92) (19). Antibodies
were incubated with nuclear extract 60 min prior to the addition of
probe.
The appearance of ISGF3
in response to IFN- can be demonstrated by an electrophoretic
mobility shift assay with a radiolabeled ISRE oligonucleotide (Fig.
1A). Antibodies used in our
study that specifically recognize STAT1, STAT2, or p48 reduce the
appearance of the ISGF3·DNA complex when added to the DNA binding
reaction, demonstrating the protein presence in the complex.
The formation of ISGF3 suggested that the STAT1 and STAT2 molecules
were unique in their ability to associate with the p48 subunit. It was
possible that STAT1 and STAT2 could only associate with the p48
molecule in conjunction with the DNA target. Alternatively, interaction
with the p48 molecule might require an IFN--induced STAT1·STAT2
heterodimer or an IFN-
-induced modification of STAT1 or STAT2.
Several models of protein-protein interactions were feasible including
the possibility that p48 could discriminate between the STATs and
specifically interact with one of the STAT molecules before and/or
after IFN-
stimulation.
To determine the specificity of p48 involvement in the formation of the
ISGF3 transcription factor we analyzed association of p48 with the
STATs by coimmunoprecipitation of protein lysates prepared from
mammalian cells. Cells were either untreated or stimulated with IFNs,
and proteins in the lysates were assayed by immunoprecipitation,
SDS-PAGE, and immunoblot analysis. A specific interaction of p48 with
the STAT2 molecule was demonstrated both prior and subsequent to
IFN- stimulation (Fig. 1B). Coimmunoprecipitation of the
STAT2 molecule with anti-p48 antibodies showed a preexisting association of these proteins (lane 1) which increased
following IFN treatment (lanes 2 and 3). Extended
treatment with IFN-
is known to increase the steady-state levels of
p48 protein, and for this reason there is an increase in the level of
preexisting STAT2·p48 detectable in these assays. Anti-STAT2 antibody
reciprocally is able to coimmunoprecipitate the p48 molecule
(lane 4).
What is clearly noted is that there is no association detectable
between p48 and STAT1, whether anti-p48 antibodies or anti-STAT1 antibodies are used (lane 5). However, the ability of STAT1
to coimmunoprecipitate with STAT2 after IFN- treatment is readily apparent, as is the IFN-
-induced phosphorylation indicated by a
decreased STAT1 mobility (lane 4) (7). A cell line that is deficient in the STAT1 protein (U3A; Ref. 20) was used to demonstrate that the STAT2·p48 complex formation is independent of the presence of STAT1 (Fig. 1C).
Since the STAT2·p48
complex exists in untreated cells, the protein interaction is not
dependent on protein modification induced by IFN-. For this reason
we analyzed the association of STAT2 and p48 in vitro to
determine if the interaction required an unidentified bridging molecule
(Fig. 2). An in vitro
transcription/translation system was used to generate protein
corresponding to STAT2 or STAT1 radiolabeled with
[35S]methionine. Radiolabeled proteins were incubated
with bacterially expressed GST-p48 protein or GST protein immobilized
on glutathione-agarose beads. Bound proteins were eluted, separated by
SDS-PAGE, and detected by autoradiography. The protein-protein
associations mimicked those seen in vivo. A specific
in vitro interaction of STAT2 with p48 was readily
detectable, whereas the STAT1 protein did not interact with p48. These
results demonstrate a direct interaction of STAT2 with p48 that does
not require a bridging or docking molecule and does not require an
IFN-induced modification.
Hybrid STAT Molecules Map p48 Recognition Site on STAT2
To
identify the region of STAT2 that specifically interacts with p48
in vivo we generated hybrid molecules of STAT1 and STAT2. Since the STAT molecules have similarity in domain organization and
amino acid sequence this approach should maintain integrity of the STAT
structure. The STAT chimeras used in the assays are fusions at
324aa/325aa, and therefore the carboxyl-terminal region of the proteins
contain the DNA binding specificity region, the SH2 domain, and the
phosphotyrosine domain of one specific STAT molecule (21-23) (Fig.
3A). The ability of p48 to
interact with these chimeric proteins was tested in vivo and
in vitro.
Protein lysates were prepared from cells stably transformed with the
STAT2/1 chimera or the STAT1/2 chimera and analyzed for association
with p48 by coimmunoprecipitation/immunoblot. The STAT2/1 chimera
encodes a protein of approximately 91 kDa similar to STAT1. It is
identified by immunoprecipitation with an antibody that recognizes the
carboxyl terminus of STAT1 and by immunoblot with an antibody that
recognizes the amino terminus of STAT2. The STAT2/1 chimera was found
to interact specifically with p48 by coimmunoprecipitation with
anti-p48 antibodies (Fig. 3B, lanes 1 and
2). Immunoprecipitation of STAT2/1 with anti-STAT1C
antibodies demonstrates a detectable signal for p48 (lane
3). The middle panel shows a longer exposure of the p48
section of the film. The expression levels of STAT2/1 are lower than
the endogenous STAT2 and may be responsible for the lower levels of
detectable p48. It is also possible that the carboxyl terminus of STAT2
can contribute to the stability of associated p48. It can also be noted
that a doublet of STAT2/1 appears after IFN- treatment. The slower
migrating band corresponds to tyrosine-phosphorylated STAT2/1 as can be
shown by reaction with anti-phosphotyrosine antibody (lower
panel).
Although association of STAT2 with p48 does not require STAT1 (Fig. 1), it was possible that the association of the STAT2/1 chimera with p48 was dependent on the function of an endogenous STAT2 molecule. To exclude this possibility, cells were analyzed which are deficient in the endogenous STAT2 protein (U6A; Ref. 24) (Fig. 3C). These cells were transformed stably with the STAT2/1 hybrid gene. Protein lysates were prepared and tested by immunoprecipitation/immunoblot for STAT2/1 interaction with p48. The analysis clearly demonstrated the ability of the p48 antibody to coimmunoprecipitate STAT2/1 in the absence of the STAT2 molecule.
The ability of the reciprocal hybrid, STAT1/2, to bind p48 was also tested. Stable transformants were generated with a STAT1/2 chimera that contained a T7 epitope tag in the carboxyl terminus, and the specific STAT1/2 chimera was identified by reactivity with anti-T7 antibody (Fig. 3D). The STAT1/2 hybrid was not able to associate with the p48 molecule by coimmunoprecipitations. Together the experiments with STAT1/2 and STAT2/1 identified an amino-terminal 324aa region of STAT2 which was required for p48 recognition in vivo.
To determine if the STAT1/2 or the STAT2/1 protein could recognize p48
directly, an in vitro association assay was performed. Radiolabeled STAT chimera proteins were synthesized in vitro
and incubated with the GST-p48 fusion protein (Fig.
4). The STAT2/1 hybrid protein
demonstrated a specific binding to GST-p48 (lane 3) compared
with GST alone (lane 5). However, the STAT1/2 hybrid protein
did not display specific binding to the p48 protein since the levels of
protein associated with GST-p48 or GST were equivalent (lanes
4 and 6). These results confirm the in vivo
results of association of p48 specifically with STAT2/1.
Predicted Coiled Coil Domains of STAT2
The amino acid
sequences of the STAT molecules were examined for predicted structural
features that might allow a better understanding of specific
protein-protein interactions. A structural feature that was readily
identified by a statistical analysis is that of a coiled coil in the
domain of STAT2 interaction with p48 (Fig. 5) (25-27). Coiled coils are
superhelices of two or more -helices wrapped around each other and
were first recognized in many fibrous structural proteins. The first
identification of a coiled coil structure in a transcription factor led
to its functional demonstration in dimerization (leucine zipper) (28).
Two computer-based methods to detect the repetitive hydrophobic and
hydrophilic amino acids in coiled coils were used to analyze the
structure of STAT2 (Coils, 26; Paircoil, 27) (Fig. 5). Both methods
predicted two coiled coils within the region necessary to bind p48:
Coils 138-173 and 192-231 amino acids; Paircoil 138-173 and
191-229. An analysis of the STAT1 molecule also revealed predicted
coiled coils within this region: Coils 134-186 and 255-297 amino
acids; Paircoil 131-171 and 257-287. Since only the STAT2 molecule
appears to bind p48, the sequence and/or the spacing of the STAT2 coils
may be critical.
A STAT2 Domain That Associates with p48
The functional domain
of STAT2 which interacts with p48 was further defined by constructing a
STAT1 chimera encoding a substitution of STAT2 148-324aa for the
homologous region of STAT1 (Fig.
6A). The ability of this
STAT1/2/1 molecule to interact with p48 was seen following transfection
into U3A cells.2 The ability
of the STAT1/2/1 protein to bind p48 was also demonstrated in
vitro (Fig. 6B). Radiolabeled STAT1/2/1 was synthesized
in vitro and incubated with the GST-p48 fusion protein or
the GST protein. The STAT1/2/1 molecule specifically associated with
the GST-p48 protein. The STAT1/2/1 molecule has a region of STAT2 that
contains the two predicted coiled coils. However, the amino-terminal junction of STAT1/2/1 occurs between STAT1 at 147aa and STAT2 at 148aa,
and therefore part of the first predicted coiled coil of STAT2 contains
a substitution with the STAT1 coiled coil (134-147aa) (Fig. 5).
Nevertheless, the STAT1 substitution maintains a predicted coiled coil
positional start site similar to the native STAT2 region. The second
predicted coiled coil of STAT1/2/1 consists entirely of STAT2 and
therefore the spacing of the two coiled coil domains in STAT2 is
maintained.
The analyses with hybrid STAT molecules served to minimize disruption of the STAT molecule integrity, and results with the chimeras mapped a region of STAT2 interaction with p48 requiring STAT2 148aa to 324aa. Although chimeras were used, it was possible that an amino-terminal fragment of STAT2 was sufficient to interact directly with p48. To test this possibility several fragments of the STAT2 protein were assayed for binding to p48 in vitro. The in vitro transcription/translation system was used to generate radiolabeled STAT2 protein fragments from the native STAT2 gene or from STAT2 deletion mutants. The DNAs were linearized with different restriction enzymes prior to transcription to produce the different proteins that were tested for binding to GST-p48 (Fig. 6A). The results of these binding assays demonstrated that a STAT2 protein fragment containing 1-324 amino acids (SphI), as is present in the STAT2/1 chimera, was sufficient to bind p48 (Fig. 6B). However, a STAT2 protein fragment of 1-125 amino acids (ApaI) did not bind p48. These analyses confirmed the in vivo binding results.
To define further the carboxyl-terminal border of STAT2 necessary to bind p48, a series of deletion constructs was generated with Bal-31 exonuclease initiating at the Bsu36I site (270aa). These constructs were used to generate STAT2 protein fragments for the in vitro binding assay corresponding to 1-190, 1-217, 1-230, and 1-250 amino acids (Fig. 6A). The STAT2 proteins encoding 1-250 and 1-230 amino acids contain both of the predicted coiled coils and specifically bind p48 (Fig. 6C). However, deletion mutations that interrupt or remove the second predicted coiled coil and possess only the first coiled coil do not recognize the p48 molecule (1-217 and 1-190 amino acids). These results show a requirement for the second predicted coiled coil of STAT2 for binding to p48.
This study provides evidence that a molecular interaction
preexists between STAT2 and p48 before and after IFN- stimulation. Although the STAT molecules share a general structural similarity and
significant amino acid identity, they appear to possess distinct elements that allow selective interactions with diverse transcription factors (29, 30). The multimeric ISGF3 transcription factor formed in
response to IFN-
contains STAT1 (91- or 84-kDa splice products),
STAT2 (113 kDa), and p48. All three protein subunits are required for
specific ISRE DNA binding and transcriptional activation. The formation
of ISGF3 was shown previously to require STAT1 and STAT2 tyrosine
phosphorylation, dimerization, and translocation to the nucleus (3). It
was proposed that the STAT1·STAT2 dimer subsequently associated with
the p48 subunit on the ISRE DNA target to form ISGF3 (31, 32). We show
here that a specific interaction of the STAT2 molecule with p48 exists
in the absence of the DNA target and appears to determine formation of
ISGF3. Association of p48 with STAT1 was not demonstrable by
coimmunoprecipitations of cell lysates or by in vitro
binding assays. Although the IFN-
-induced STAT1·STAT2 heterodimer
was detected readily by coimmunoprecipitation with anti-STAT1 antibody,
the dimer did not contain p48 (Fig. 1B, lane 5).
These results are interpreted to indicate the existence of two distinct
complexes following IFN-
stimulation: STAT2·p48 and STAT2·STAT1.
Association of the bipartite complexes appears to occur on the DNA ISRE
target to form the multimeric ISGF3 transcription factor. The exact
stoichiometry of components in ISGF3 is as yet unresolved. The
estimation of relative subunit levels following purification of ISGF3
by DNA affinity (9, 33) and the ability of p48 and STAT1 in ISGF3 to
contact the ISRE (32) are compatible with our results and model. The
configuration of direct half-sites in the ISRE may allow STAT2·p48 to
bind to one half-site and STAT2·STAT1 to bind to the other half-site.
Alternative models have also been envisioned (32), but in the absence
of additional structural information the models remain to be
tested.
The amino-terminal domain of STAT2 that associates with p48 appears to be defined by a structure containing two predicted coiled coils at 138-173 and 191-231aa (Fig. 5) (25-27). Although STAT1 also has predicted coiled coils in this region, the specific amino acid sequence or domain structure does not specify interaction with p48. Previous studies had suggested an association of STAT1 and p48 (31, 34). A directed yeast two-hybrid transcription system detected interaction of STAT1 with p48 (31), and in vitro mixing of crude lysates from cells overexpressing STAT1 and p48 showed apparent complex binding to DNA (38). In contrast, our studies performed in human cells at physiological levels of proteins only detect STAT2·p48 complexes. Our studies in vitro demonstrate a specific interaction of p48 with a STAT2 domain encompassing the two predicted coiled coils between 138 and 230 amino acids. The STAT2-p48 interaction is resistant to 0.5% Nonidet P-40 since this detergent was included in the lysis buffer. Even if lysates are prepared in the absence of detergent the STAT2·p48 complex is apparent, but there is no detectable p48 association with STAT1. A previous report demonstrated in vitro binding of p48 with both STAT2 and STAT1 using a similar GST interaction assay (31). The data clearly showed a p48 interaction with STAT2 which was many fold greater than that detectable with STAT1. Certainly there are inherent differences in amounts of 35S-radiolabeled proteins synthesized in vitro that could affect the results, as well as differences in binding/washing conditions.
Cells express many different STAT factors, and accurate STAT activation and function in response to cytokines depend on precise protein-protein interactions. Recruitment by specific cytokine receptors and Janus kinases can dictate distinct STAT activation. Association of STATs with heterologous transcription factors can determine transcriptional induction of particular genes. The specificity of STAT2 interaction with p48 appears to dictate recognition of the tandem GAAA repeat of the ISRE by the ISGF3 complex. A region of p48 which interacts with the STATs to form ISGF3 was originally identified by in vitro reconstitution experiments to be located in the carboxyl-terminal half of the protein, 217-377aa (35). The p48 protein does not contain regions of predicted coiled coil, but it does contain pockets of hydrophobic stretches in the carboxyl terminus which may participate in interaction with STAT2. Transcriptional specificity is regulated by precise protein-protein interactions acting in conjunction with a particular DNA target site, and it is the precision of the molecular interactions that confers specificity of a rapid and specific biological response to distinct extracellular stimuli. Elucidation of the precise amino acid interactions that drive STAT2·p48 complex formation can contribute to our understanding of other protein-protein associations and offer a target of activation/intervention of receptor to nucleus signaling.
We thank all of the members of the laboratory for support, especially Carrie Mahlum for assistance with plasmid constructions. We thank James E. Darnell, Jr., for the STAT1 and STAT2 cDNAs, David E. Levy for the p48 cDNA, and George Stark and Ian Kerr for the cells deficient in STAT1 (U3A) and STAT2 (U6A). IFNs were a gift from Hoffman-LaRoche Inc.