(Received for publication, April 15, 1997, and in revised form, June 16, 1997)
From the Fragments of characteristic size retaining the
ability of sequence-specific DNA binding were generated by partial
proteolysis of transcription factor Stat3 with trypsin, chymotrypsin,
or Staphylococcus V8 proteinase. The molecular masses of
the smallest DNA-binding fragments were 75, 48, and 32 kDa after
digestion with V8 proteinase, chymotrypsin, and trypsin, respectively.
The fragments contained major parts of the domain controlling the
sequence specificity of DNA binding (amino acids 406-514), the SH3 and
SH2 domains, and the phosphorylated tyrosine residue Tyr-705, but not
the C-terminal 20 amino acids. The N terminus of the 32-kDa tryptic
fragment (ANCDASLIV) matched the sequence of amino acids 424-432
deduced from cDNA. The fragments were observed after proteolytic
treatment of preformed complexes between DNA and native factors eluted
from rat liver nuclei or recombinant, tyrosine-phosphorylated rat Stat3 from insect cells. It was possible to elute all three minimal fragments
from their complexes with DNA and to obtain specific re-binding. The
minimal fragments eluted from complexes with DNA still contained the
phosphorylated Tyr-705 and the SH2 domain suggesting that they were
probably bound to DNA as dimers. The DNA-binding domain of Stat3
identified by these experiments overlapped the domain previously
identified by genetic experiments as the domain controlling the
sequence specificity of DNA binding. The DNA-binding domain defined
here by partial proteolysis probably represents an autonomously folding
portion of Stat3.
Stat3 is a transcription factor mediating the effects of a variety
of cytokines including interleukin 6 (IL6)1 and IL6-related
cytokines on the transcriptional induction of their target genes
(1-6). In particular, Stat3 is the factor primarily responsible for
the cytokine-mediated induction of class II acute phase genes during an
acute phase response of the liver (1, 7-15). In the absence of
cytokine signals, Stat3 is present as a functionally latent monomer in
the cytoplasm. In response to cytokine signals Stat3 is phosphorylated
at a single tyrosine (Tyr-705) by receptor-associated JAK/TYK kinases.
This process, referred to as the "activation" of Stat3, leads to
the dimerization of monomers via their phosphotyrosines and SH2 domains
in an antiparallel orientation and to the translocation of dimers to
the nucleus (2-5, 16, 34). An analogous process leads to the formation of heterodimers, for example between Stat1 and Stat3 (4). Stat3 dimers
bind at specific response elements in the control regions of their
target genes, including the class II acute phase gene Apart from their SH2 domains Stat factors have no sequence homology
with other known transcription factors, and therefore their DNA-binding
domains, transcriptional transactivator domains, and nuclear
localization domains are of unknown types. This situation presents a
rare opportunity to analyze a novel type of DNA-binding domain. The
position of the DNA-binding domain within Stat3 is not known with
precision, but it cannot reside in the C-terminal 40 amino acids,
because these are dispensable for DNA binding. Attempts to confine this
domain by construction of mutants with progressive deletions extending
inward from the N and C termini have met with limited success. As soon
as deletions from the C terminus extended beyond Tyr-705, efficient DNA
binding was abolished, because dimerization apparently is a
prerequisite for high affinity DNA binding. Similarly, mutants in which
Tyr-705 was replaced by a different amino acid have been constructed.
The mutant factors had lost the ability to dimerize and to bind DNA
(4). It is not formally excluded that Stat factor monomers may be able
to attach to their specific DNA-binding sequences with very low
affinity. However, if this binding mode existed, it must have gone
undetected by the methods used to date. Deletions from the N terminus
have also been of limited use, because the corresponding mutant factors were not efficiently phosphorylated. The N terminus of Stat factors is
important for docking to the receptor-JAK/TYK kinase complex, and this
in turn is essential for the phosphorylation of Stat factors (4). Thus,
the genetic approach to map the DNA-binding domain of Stat factors has
been of limited use.
A region controlling the specificity of DNA binding has been identified
by domain-exchange experiments, so-called "domain swap"
experiments, between Stats 1 and 3 or Stats 1 and 6 (18, 23). The
region located between amino acids 406 and 514 in Stat3 has been mapped
by this approach. However, it was not clarified whether this region
contained the actual DNA-contact domain of Stat3 or whether it
represented only a domain controlling the specificity of DNA binding
without entering into direct contact with DNA.
To overcome these difficulties and to identify the DNA-contact domain
of Stat3, we have relied on an approach including the initial tyrosine
phosphorylation of the intact molecule and the subsequent removal by
partial proteolysis of all parts that were no longer required for DNA
binding once tyrosine phosphorylation had been achieved. This approach
has allowed us to define a fragment of approximately one-third the size
of Stat3 that was still capable of sequence-specific DNA binding. Here
we describe the delineation and immunological characterization of this
fragment.
Nuclear extracts from rat
livers were prepared and electrophoretic mobility shift assays (EMSA)
were performed as described previously (17, 24-26). The radiolabeled,
double-stranded oligonucleotide TB2 used in most DNA-binding reactions
carries two copies of the core site of the IL6 response element of the
rat Protein-coding
fragments of rat Stat3 cDNA were inserted into the bacterial
expression vector pET15b (Novagen) generating fusion polypeptides with
an N-terminal hexahistidine (6xHis)-tag. Construction was
verified by sequence analysis, and the constructs were tested for
expression in Escherichia coli BL21 (Novagen) after
induction with 1 mM isopropylthiogalactoside. Recombinant proteins were purified under denaturing conditions using a nickel nitrilotriacetic acid column (Quiagen) following the instructions of
the manufacturer and dialyzed against phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4). Rabbits and mice were separately
immunized with approximately 500 and 50 µg, respectively, of the
purified recombinant Stat3 fragments. Immunization, booster injections,
collection, and processing of the sera were performed following
standard procedures (31) as a commercial service by Charles River
Laboratories, Kisslegg, Germany. The fragments used were EX18 (amino
acids 1-159 of the rat Stat3 cDNA sequence; Ref. 17), RD7 (amino
acids 231-334), PII.1/10 (amino acids 405-555), and SD4 (amino acids
604-770). The anti-SD4 serum was absorbed with E. coli
proteins. To this effect, an insoluble acetone precipitate from
E. coli BL21 cells was incubated overnight at 4 °C with
the serum. The precipitate was then removed by centrifugation, and the
supernatant represented the absorbed serum.
Western blot experiments were
performed after electrophoresis of protein extracts in SDS containing
17.5% polyacrylamide gels (31, 32). For the verification of the
specificity of polyclonal rabbit sera 20 µg of crude extract from
isopropylthiogalactoside-induced E. coli BL21 bacteria
expressing Stat3 fragments were used. For the analysis of the minimal
DNA-binding domains of Stat3, the eluted fraction representing 50 µg
of input crude extract from insect cells was loaded on the gel. The
proteins were transferred to a BA-85 nitrocellulose membrane
(Schleicher & Schuell). Membranes were blocked with 3% bovine serum
albumin in Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 7.5) and probed with the anti-Stat3 antibodies
generated in this study (mouse anti-RD7, mouse anti-PII.1/10, and
rabbit anti-SD4) or with the commercial antibodies rabbit anti-C20
(Santa Cruz), mouse anti-S3-N (Transduction Laboratories), and the
anti-phosphotyrosine antibody 4G10 (Upstate). Goat anti-rabbit IgG
coupled to horseradish peroxidase (Sigma) and goat anti-mouse IgG
coupled to horseradish peroxidase (Dianova) were used as second antibodies. Signals were detected using the enhanced chemiluminescence system (Amersham Corp.).
Five
hundred pmol of biotinylated, double-stranded oligonucleotide JR2
(consisting of one JR1 and one TB1 binding site in tandem)2
was allowed to react with 1 mg of streptavidin coupled to magnetic porous glass beads (Controlled Pore Glass, CPG Inc., Lincoln Park, NJ)
according to the manufacturer's protocol. Crude extract (250 µg)
from Sf21 insect cells coinfected with baculoviral expression constructs for rat Stat33 or
murine JAK2 kinase (from Drs. S. McKnight and U. Schindler), respectively, and harvested 50 h postinfection were incubated with
the JR2 oligonucleotide coupled to magnetic porous glass beads. Binding
was allowed to proceed for 10 min at 25 °C in 500 µl of CP buffer
(10 mM Hepes, pH 7.6, 0.1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol, 6 mM
MgCl2, 1.25 mM CaCl2, 25 µg of
salmon sperm DNA) supplemented with 50 mM KCl (17, 24).
Subsequent digestion of DNA-bound Stat3 was performed for 10 min at
25 °C with trypsin or chymotrypsin and at 37 °C with V8
proteinase in 500 µl of CP buffer supplemented with 100 mM KCl. Trypsin (14 units), chymotrypsin (8 units), and V8
proteinase (375 units) were used to treat 250 µg of crude insect cell
extract containing recombinant rat Stat3. After completion of the
reaction the beads were separated magnetically and washed twice with CP
buffer supplemented with 100 mM KCl. DNA-bound proteins
were eluted with 200 µl of CP buffer supplemented with 600 mM KCl and 2 µg of bovine serum albumin. For EMSA assays,
the eluted protein was used directly. For Western blot analysis eluted
proteins were precipitated with trichloroacetic acid and resuspended in
SDS sample buffer (32).
Ten mg of crude
extract from Sf21 insect cells coinfected with baculoviral expression
constructs for Stat3 and JAK2 and harvested 50 h after infection
were incubated with the double-stranded biotinylated JR2
oligonucleotide bound to streptavidin/magnetic porous glass beads.
Tryptic digestion of the DNA-bound Stat3 was performed as described
above, and the eluted minimal fragment was electrophoretically separated in an SDS-polyacrylamide gel. The relevant fragment was
transferred to a Glassybond membrane (Biometra) as described previously
(33). N-terminal amino acid sequencing was performed by automated Edman
degradation using an Applied Biosystems 492A sequencer.
Proteolytic digestion of rat Stat3 produces defined minimal
fragments capable of sequence-specific DNA binding. Nuclear protein extracts from rat livers excised 6 h after induction of an
experimental acute phase response were used as the initial source of
activated Stat3. Earlier studies had demonstrated that Stat3 was the
most abundant activated Stat factor in liver nuclei at this stage of the acute phase response. At this time activated Stat1 was present in
negligible concentrations and activated Stat5 was undetectable in the
nuclei (17). Protein-DNA complexes with the radiolabeled double-stranded oligonucleotide probe were first assembled in vitro and then treated with increasing concentrations of either trypsin, chymotrypsin, or Staphylococcus V8 proteinase. In
all three cases defined complexes were produced that migrated as
focused bands in electrophoretic mobility shift experiments (EMSAs)
even after digestion with elevated concentrations of the proteinases (Fig. 1). The treated complexes migrated
faster than the untreated complex (complex II) indicating a discrete
loss of protein mass. DNA binding of the minimal Stat fragments
contained in these complexes was still sequence-specific as shown by
competition gel mobility shift experiments (Fig.
2). Addition of an excess of
nonradioactive oligonucleotides TB1 or CA1, representing efficient Stat
factor binding sites (see "Experimental Procedures") to the
DNA-binding reaction prior to addition of the proteinases prevented
formation of the faster migrating complexes. Addition of an excess of
the mutant oligonucleotide mTB1, an ineffective Stat factor binding site, had no effect. Thus, fragments of Stat3 that remained bound to
DNA after proteolytic digestion of preformed complexes remained bound
with the same sequence specificity as intact Stat3.
When nuclear protein extracts from acute
phase rat livers were first treated with proteinases and assembly of
complexes with DNA was attempted subsequently, no more complexes were
observed after treatment with V8 proteinase (Fig. 2B).
Faster migrating complexes of the same characteristic mobility as in
Fig. 2A were also observed after treatment with trypsin and
chymotrypsin. These findings suggest the existence of cleavage site(s)
for V8 proteinase within the minimum DNA-binding fragment accessible to
the proteinase in free Stat3 but not in complexes between Stat3 and its
DNA target. By contrast, treatment of free Stat3 with limited amounts
of trypsin and chymotrypsin generated fragments capable of
sequence-specific DNA binding. The sequence specificity of DNA binding
of the proteolytic fragments was established by competition gel
mobility shift experiments with appropriate oligonucleotides (Fig.
2B).
For an immunological identification of the portion of Stat3
contained in the minimal DNA-binding fragments, a set of 7 overlapping fragments, each approximately 100 to 200 amino acids in length, were
expressed in E. coli (Fig. 3).
Together these fragments covered the complete Stat3 sequence. Fragments
EX18, RD7, PII.1/10, and SD4 were purified and used to immunize rabbits
and mice. The N-terminal fragment EX18 was not immunogenic in our
experiments. Therefore, the two commercial antibodies S3-N and C20,
directed against N- and C-terminal portions of Stat3, respectively,
were included in this study. The specificity of the antisera was
verified in Western blot experiments with bacterial extracts expressing
the fragments used for immunization (Fig.
4). All antibodies specifically reacted
only with fragments carrying the epitopes used to generate them and
were therefore suitable to map the minimal DNA-binding fragments of
Stat3.
Further structural
characterization of the minimal DNA-binding fragments required the
availability of Stat3 in semi-preparative quantities in the 0.1-1 nmol
range, with 1 nmol equalling approximately 90 µg. To this effect
recombinant rat Stat3 and murine JAK2 kinase were produced from
baculoviral expression constructs in insect cells (see "Experimental
Procedures"). Double infection of insect cells with both of these
constructs produced correctly tyrosine-phosphorylated, specifically
DNA-binding rat Stat3 in the required
quantities.4 For purification
of the minimal DNA-binding fragments on the semi-preparative scale,
proteolytic digestions were performed on Stat3-DNA complexes. For this
purpose the biotinylated double-stranded synthetic oligonucleotide JR2
carrying an efficient Stat3 binding-site was used as the DNA target
(see "Experimental Procedures"). Tyrosine-phosphorylated Stat3
contained in insect cell extracts was allowed to bind to this target
and enriched with streptavidin-coated magnetic beads (see
"Experimental Procedures"). The beads were then collected, washed,
and treated with the proteinase of choice. After completion of the
reaction, the beads were again collected and washed, and the minimal
DNA-binding fragments were eluted. The recovered fragments were then
used for further analysis in EMSA experiments or for electrophoresis in
SDS-polyacrylamide gels and Western blots.
Full-length
Stat3 and its minimal DNA-binding fragments generated by treatment with
the three proteinases were analyzed for their ability to re-bind DNA in
EMSA experiments (Fig. 5). Re-binding was
assayed using the radiolabeled synthetic oligonucleotide JR1. Stat3
from insect cell extracts and eluted Stat3 formed a complex III of
standard mobility, consisting of one Stat3 dimer (Fig. 5, tracks
2 and 3). The eluted and re-bound minimal tryptic and chymotryptic fragments generated complexes migrating with the same
increased mobilities as those originally detected in Fig. 1 (Fig. 5,
tracks 4 and 5, respectively). The eluted and
re-bound V8 proteolytic fragment also generated a complex of
indistinguishable mobility as the original minimal complex shown in
Fig. 1. Re-binding of the eluted V8 minimal fragment was
sequence-specific as shown by competition gel shift experiments with
appropriate competitors (Fig. 5B).
To identify the portions of Stat3 contained in the
minimal DNA-binding fragments, Western blot experiments were performed with the eluted fragments (Fig. 6). The
antibodies PII.1/10, SD4, and the anti-phosphotyrosine antibody reacted
with full-length Stat3 and the minimal DNA-binding fragments generated
by all three proteinases. These fragments had approximate molecular
masses of 32, 48, and 75 kDa for the tryptic, chymotryptic, and V8
fragments, respectively (Fig. 6A). The C20 antibody reacted
only with full-length Stat3 (Fig. 6B). The RD7 antibody
reacted with full-length Stat3 and the minimal chymotryptic and V8
fragments but not with the 32-kDa minimal tryptic fragment. The S3-N
antibody was reactive only with full-length Stat3 and the minimal V8
fragment. By comparison with the epitope map (Fig. 3), we concluded
that the 32-kDa minimal tryptic fragment still contained the domain
controlling the specificity of DNA binding (amino acids 406-514), the
SH2 domains, and Tyr-705. It did not contain the C-terminal 20 amino
acids carrying the C20 epitope and also lacked the first 300-400 amino
acids, carrying the RD7 and S3-N epitopes (Fig.
7).
The minimal
tryptic DNA-binding fragment of Stat3 was prepared by elution from DNA
bound to magnetic beads on the semi-preparative scale. The eluted
fragment was electrophoretically separated in an SDS-polyacrylamide
gel, and the relevant 32-kDa band was excised. The excised protein was
transferred to a membrane and subjected to N-terminal amino acid
sequence analysis (see "Experimental Procedures"). A sequence of 9 amino acids (ANCDASLIV) was obtained, matching amino acids 424-432 of
the published rat Stat3 sequence deduced from cDNA (Ref. 17; Fig.
8). The C terminus of the fragment has
not been determined by direct structural analysis. If the molecular
mass of 32 kDa is reliable, then the C terminus should be located at
one of the following three amino acid positions: Lys-707, Lys-709, or
Arg-729 (Fig. 8). If the C terminus were located at Lys-707, then the
calculated molecular mass of this fragment would be 32.3 kDa. If it
were located at Lys-709 or Arg-729, then the molecular masses would be
32.4 or 34.6 kDa, respectively.
The main new results and conclusions drawn from this study were as
follows. 1) Digestion of rat Stat3 with trypsin, chymotrypsin, and
Staphylococcus V8 proteinase generated minimal DNA-binding fragments of characteristic sizes (32, 48, and 75 kDa, respectively). 2) The minimal fragments generated by proteolytic digestion of both
free and DNA-bound Stat3 retained the ability of sequence-specific DNA
binding. 3) Minimal DNA-binding fragments of recombinant Stat3 eluted
from DNA were capable of sequence-specific re-binding. 4) Minimal
DNA-binding fragments of Stat3 contained the SH2 domains and were
tyrosine-phosphorylated. They did not include the extreme N- and
C-terminal portions of Stat3. 5) The minimal DNA-binding fragments also
contained the region previously described by other authors as the
region controlling the sequence specificity of DNA binding (amino acids
406-514), but the fragment generated by tryptic digestion did not
contain amino acids 406-423.
The observation of a minimal DNA-binding domain of Stat3 generated by
digestion with three different proteinases suggests that Stat3 contains
a localized DNA-contact domain, which is both necessary and sufficient
for specific DNA binding. Hence, in this respect Stat3 is similar to
many other eukaryotic DNA-binding proteins that contain functionally
autonomous DNA-binding domains. The DNA-binding function of Stat3 thus
is not generated by the combined contributions of several discontinuous
sub-domains dispersed over the entire molecule.
Interestingly, the minimal DNA-binding domain contains a cleavage site
for V8 proteinase that is protected in DNA-bound Stat3 but accessible
in free Stat3. This finding suggests that Stat3 either undergoes a
previously not recognized conformational change upon DNA binding or
that the bound DNA constitutes a sterical hindrance for the
accessibility of the cleavage site by V8 proteinase.
The competition gel mobility shift experiments (Fig. 2) document
sequence specificity of DNA binding for the minimal tryptic and
chymotryptic fragments (Fig. 6). These minimal fragments contained both
the SH2 domain and phosphorylated Tyr-705. It is therefore reasonable
to assume that they were bound as dimers, because this is the generally
accepted DNA- binding mode of intact Stat factors. However, no direct
evidence (such as protein-DNA cross-linking data or competition studies
with a phosphotyrosine peptide, Ref. 18) was provided here for a
dimeric organization of the DNA-bound minimal fragments. It is only the
simplest interpretation of our data to assume that partial proteolysis
of a DNA-bound dimer removed terminal portions of the protein not
involved in DNA binding without changing the organization of the
DNA-bound core of the dimer. At present, it is not clear whether
dimerization is an absolute prerequisite for the sequence specificity
of Stat factor binding to DNA or only a mechanism enhancing the
affinity of binding. All available evidence points to dimerization as a
requirement for high affinity binding detectable with the currently
available methods. However, weak sequence-specific binding of Stat
factor monomers below the detection threshold of current methods has not been formally excluded.
The immunological mapping of the portion of Stat3 contained in the
minimal DNA-binding fragments produced results compatible with the
mapping by partial amino acid sequence analysis. Our results are also
consistent with previous conclusions of other authors (18, 23) who
reported that the domain controlling the sequence specificity of DNA
binding is approximately located between amino acids 400 and 500. Our
results suggest that this specificity-controlling region may in fact be
identical with the DNA-contact region. However, our data do not allow
us to locate the C-terminal boundary of the minimal DNA-contact domain
with precision. This is due to the fact that the minimal binding
fragments defined here carry not only the DNA-contact domain but also
all sequences required for dimerization. We cannot decide from our data
whether the minimal DNA-contact domain ends in the vicinity of amino
acid 500 or extends beyond this region and includes sequences between
amino acids 500 and 706. This question could probably be solved by
employing an artificial mechanism of antiparallel dimerization
operating independently of the SH2 domain and phosphorylation of
Tyr-705.
Interestingly two sequence motifs previously identified by other
authors (23) as essential for the sequence specificity of binding are
included in the minimum tryptic fragment reported here. These residues,
VTEEL and SLPVVV (432-436 and 458-463 in rat Stat3), are located
close to the N terminus of the minimal tryptic fragment (position 424;
Fig. 8). This observation still does not allow us to conclude formally
that the sequences jointly contained in the region defined here and by
the genetic approach (23; amino acids 424-500 approximately) must
contain the DNA-contact domain. However, this suggestion is now very
plausible.
An alternative way to identify the minimal DNA-contact domain would
consist in mapping amino acid residues engaged in direct contact with
DNA by protein-DNA cross-linking. Such experiments are currently
underway. Finally, it should be possible to delineate the DNA-contact
region by x-ray diffraction of protein-DNA crystals. However, such
crystals and the crystal structure of free Stat3 have so far not been
obtained. One of the reasons why it may have been difficult to generate
crystals of free Stat3 probably is that this molecule consists of
several autonomously folding domains, such as the DNA-contact domain
and the transcriptional transactivator domain. These regions most
likely are connected by flexible linkers carrying the cleavage sites
for the proteinases employed here and may not assume a sufficiently
well defined position in space to allow the formation of crystals. The
fact that it was possible to define a minimum DNA-binding fragment of
only one-third the size of intact Stat3 suggests that this fragment may
represent an autonomously folding domain. The observation that this
minimal tryptic fragment was well defined and formed a single sharp
band in SDS-polyacrylamide
gels5 is a further argument
in favor of this view. If this fragment indeed represented an
autonomously folding domain, then it may be possible to crystallize it
alone or in a complex with specific DNA sequences and to obtain x-ray
diffraction data. Attempts to crystallize this region both alone and in
complex with DNA are currently underway.
We thank Dr. J. Miller (Lilly) for advice on
the construction of baculoviral expression vectors and Drs. S. McKnight
and U. Schindler (Tularik Inc., San Francisco) for providing the
baculoviral expression construct for mouse JAK2. We are grateful to Dr.
L. Miller for providing the insect cells.
Chair of Genetics,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
2-macroglobulin (
2M), and mediate their
transcriptional induction (1, 2, 8, 9, 11, 12, 15, 17).
Heterodimers often occupy the same palindromic response elements as
homodimers, because the members of the Stat factor family bind to
similar DNA target sequences. These binding sites generally have a
palindromic symmetry allowing the factors to bind in an antiparallel
orientation. Binding sites deviating from perfect palindromic symmetry
have been observed in a number of genes such as the rat
2M gene, and a set of rules have been derived specifying
sequences that are preferred binding sites for various members of the
Stat factor family. These rules describe the binding specificity as a
function of the distances between the half-sites of the palindrome, of the spacer sequences between the half-sites, and the sequences of the
half-sites (18). Stat3 dimers are capable of cooperative binding to
dimers of other Stat factors. The cooperative interaction between
dimers bound at tandem sites is mediated by the N-terminal domains of
the Stat factors (19-21). The transcriptional transactivator domain is
located in the C-terminal 40 amino acids of Stat3. Naturally occurring
C-terminally truncated molecules lack the transactivator function.
These truncated proteins still carry the DNA-binding domain and compete
with the intact molecules for the same DNA-binding sites. Thus, they
act as dominant negative inhibitors of the full-length factors (2-5,
22).
Gel Mobility Shift Experiments
2M gene (27, 28) and is a binding site of
intermediate strength for Stat3 (12, 17). Competition EMSA experiments
were performed using the nonradiolabeled, double-stranded
oligonucleotides TB1, mTB1, and CA1 in a 100-fold (TB1, mTB1) and a
50-fold molar excess (CA1), respectively. TB1 is a 24-base pair
oligonucleotide containing one copy of the core site of the IL6
response element of the
2M gene (27, 28). mTB1 is a
mutant of TB1, in which the critical hexanucleotide CTGGGA was replaced
by a permutation with identical base composition, that no longer binds
Stat factors (24, 27, 28). CA1 is a 49-base pair oligonucleotide
carrying the bipartite IL6 response element of the rat
2M gene in its authentic configuration (27, 28). JR1 is
a palindromic variant of the TB1 core site (TTCCGGGAA) and a strong
binding site for Stat3.2
Proteolytic clip-shift assays were performed as described previously for the digestion of Stat3 with trypsin and chymotrypsin (29, 30).
Digestion with Staphylococcus V8 proteinase (Sigma) was performed for 10 min at 37 °C in CP buffer (see below) with 15 units
of the proteinase per 10 µg of nuclear protein extract from rat
livers.
Fig. 1.
Digestion of Stat3 with three different
proteinases reveals a minimal DNA-binding domain. Nuclear extracts
from adult rat livers were prepared 6 h after the initiation of an
experimental acute phase response. Ten µg of extract were incubated
with the radiolabeled oligonucleotide TB2 and subsequently digested
with increasing amounts of trypsin (0-5 units), chymotrypsin (0-3
units), or Staphylococcus V8 proteinase (0-125 units).
Resulting protein-DNA complexes were analyzed by gel mobility shift
(EMSA) experiments. , no protein; II, complex II, a
complex consisting of two dimers of Stat3 bound at tandem sites in TB2;
arrowheads in the right margin, minimal
DNA-binding fragments.
[View Larger Version of this Image (51K GIF file)]
Fig. 2.
Minimal DNA-binding fragments of Stat3 show
sequence-specific binding. Ten µg of nuclear extract from livers
of adult rats prepared 6 h after induction of an experimental
acute phase response were used for each lane of this EMSA competition
experiment. Binding to the radioactive double-stranded oligonucleotide
TB2 was competed by a molar excess of the nonradioactive
double-stranded oligonucleotides TB1 (lanes 3, 7, 11, and
14) or CA1 (lanes 5 and 9) carrying
intact binding sites but not by oligonucleotide mTB1 (lanes 4, 8, 12, and 15) carrying a mutated binding site; lanes 2, 6, 10, and 13, no competitor; lane
1, no protein added. A, nuclear extract digested with
proteinases after DNA binding; B, nuclear extract digested
before DNA binding. II, complex II; arrows
labeled T, C, V8, protein-DNA
complexes after digestion with trypsin, chymotrypsin, and V8
proteinase, respectively; , no proteinase; T, trypsin;
C, chymotrypsin; V8, Staphylococcus V8
proteinase.
[View Larger Version of this Image (44K GIF file)]
Fig. 3.
Fragments of Stat3 used for the generation of
polyclonal antisera and epitopes recognized by various anti-Stat3
antibodies. Fragments of Stat3 cDNA were cloned into the
expression vector pET15b as fusions with a 6xHis-tag. The fragments
were expressed in E. coli BL21 and purified on a nickel
nitrilotriacetic acid column. Rabbits and mice were immunized with
fragments EX18, RD7, PII.1/10, and
SD4, respectively. Epitopes recognized by the two commercial
antibodies S3-N and C20 are also shown.
Shaded N-terminal region, domain for cooperative binding
between dimers of Stat factors at tandem sites; DNA, domain
controlling the specificity of DNA binding; SH2, src
homology domain 2; Y, tyrosine 705; TA, transcriptional transactivator domain.
[View Larger Version of this Image (15K GIF file)]
Fig. 4.
Specificity of anti-Stat3 antibodies.
A, 20 µg of crude extract (for EX18: 1 µg of purified
fragment) from E. coli cells expressing Stat3 fragments were
loaded for each track of a 17.5% SDS-polyacrylamide gel, and the gel
was stained with Coomassie Blue. ST, molecular weight
standard; , extract from E. coli cells not expressing any
recombinant Stat3 fragment; B-F, Western blot analysis with
the antibodies S3-N (B), RD7 (C), PII.1/10
(D), SD4 (E), and C20 (F).
[View Larger Version of this Image (50K GIF file)]
Fig. 5.
Recombinant Stat3 and its proteolytic
DNA-binding fragments eluted from DNA are capable of re-binding to
DNA. Recombinant Stat3 expressed in Sf21 insect cells was tested
for DNA binding activity in EMSA experiments using the JR1
oligonucleotide. The equivalent of 5 µg of crude extract was loaded
on each lane. A, DNA re-binding of the minimal fragments;
lane 1, no protein; lane 2, crude extract;
lane 3, Stat3 re-bound after elution; lane 4, re-bound tryptic fragment; lane 5, re-bound chymotryptic
fragment; III, complex III consisting of a single dimer of
Stat3 bound at a palindromic site. B, gel mobility shift
competition for binding of the minimal V8 fragment (V8) to
oligonucleotide TB2; 1, no competitor; 2, high
molar excess of competitor TB1; 3, mTB1; 4, CA1.
Target and competitor oligonucleotides as described under "Experimental Procedures."
[View Larger Version of this Image (39K GIF file)]
Fig. 6.
The minimal DNA-binding fragments include the
SH2 domain and are tyrosine-phosphorylated. Two hundred fifty µg
of crude extract from Stat3-expressing Sf21 insect cells were incubated with oligonucleotide JR2 coupled to magnetic beads and digested with
the proteinases as described before. As a negative control, JR2/magnetic beads were incubated with proteinases in the absence of
added crude extract. The purified proteins were precipitated with
trichloroacetic acid, resuspended in SDS sample buffer, and electrophoresed in a 17.5% SDS-polyacrylamide gel. Proteins were blotted on a nitrocellulose membrane and incubated with the anti-Stat3 antibodies shown in the left margin. Detection was done with
the enhanced chemiluminescence system (Amersham Corp.). A,
anti-PII.1/10 (1:2500), anti-phosphotyrosine (1:2000), anti-SD4
(1:10000); B, anti-S3-N (1:10000), anti-C20 (1:5000),
anti-RD7 (1:2000). Arrows pointing to S3, full-length Stat3;
T, tryptic fragment; C, chymotryptic fragment;
V8, V8 fragment. + and symbols in
top row, reactions with or without added Stat3.
[View Larger Version of this Image (36K GIF file)]
Fig. 7.
Composition of the minimal DNA-binding
fragments as determined by immunological analysis. S3-N, RD7,
PII1./10, SD4, and C20 indicate the epitopes recognized
by the antibodies used. 75-, 48-, and 32-kDa fragments represent the
minimal fragments generated by digestion with V8 proteinase,
chymotrypsin, and trypsin, respectively. DNA, domain
controlling specificity of DNA binding (23). SH2, SH2
domain; Y, Tyr-705; TA, transcriptional
transactivator domain. Black box at N terminus, domain for
cooperative interaction of Stat3 dimers bound at tandem sites.
[View Larger Version of this Image (25K GIF file)]
Fig. 8.
Sequence of the minimal DNA-binding tryptic
fragment of Stat3. Double underscored sequence, N-terminal
amino acid sequence of the minimal tryptic fragment as determined by
partial amino acid analysis. *, possible C-terminal residues of the
32-kDa tryptic fragment. , Tyr-705.
[View Larger Version of this Image (42K GIF file)]
*
These studies were supported by Research Grant Hi 291/5-2
TP2 from the Deutsche Forschungsgemeinschaft (to G. H. F.) and a fellowship from the Deutsche Forschungsgemeinschaft Training Grant (Graduiertenkolleg) GRK40 (to S. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Travel support was provided by the Wallner Foundation.
To whom correspondence should be addressed: Chair of Genetics,
University of Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany. Tel.: 49-9131-85-8494; Fax: 49-9131-85-8526.
1
The abbreviations used are: IL6, interleukin 6;
2M,
2-macroglobulin; EMSA,
electrophoretic mobility shift assays.
2
J. Ripperger and G. H. Fey, unpublished
data.
3
S. Fritz and G. H. Fey, unpublished
data.
4
S. Fritz, unpublished data.
5
B. Dreier, unpublished data.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.