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(Received for publication, November 27, 1995, and in revised form, February 28, 1996)
From the Department of Pulmonary Diseases, University Hospital
Utrecht, Utrecht, The Netherlands, and the ABSTRACT The 89-kDa STAT3 protein is a latent
transcription factor which is activated in response to cytokines
(interleukin (IL)-5 and -6) and growth factors (epidermal growth
factor). Binding of IL-5 to its specific receptor activates JAK2 which
leads to the tyrosine phosphorylation of STAT3 proteins. Here we report
the cloning of a cDNA encoding a variant of the transcription
factor STAT3 (named STAT3 INTRODUCTION Stimulation of transcription factors by cytokines or growth
factors is an important step in activating specific gene transcription
leading to cell growth, differentiation, and many other cellular
functions. To activate or repress transcription, transcription factors
must be located in the nucleus, bind DNA, and interact with the basal
transcriptional machinery. Most of these processes are achieved by
phosphorylation of a transcription factor by protein kinases (1, 2).
One of the earliest signaling events after cytokine stimulation is the
activation of non-receptor protein tyrosine kinases, such as members of
the Src and Janus kinase (JAK) families (3). Many individual cytokine
receptors are linked to specific members of the JAK family which are
activated after ligand binding. The activated JAK kinases phosphorylate
and activate a novel family of transcription factors termed signal
transducers and activators of transcription
(STATs)1 (4). STAT proteins were first
recognized in the interferon- Cytokines such as interleukin-3 (IL-3), IL-5, and
granulocyte-macrophage colony stimulating factor (GM-CSF) play an
important role in hematopoiesis (23, 24, 25). Although IL-3 and GM-CSF also
have effects on other hematopoietic lineages (25, 26), the actions of
IL-5 in humans are restricted to eosinophils and basophils, since the
IL-5 receptor (IL-5R) is only expressed on these cell types (27, 28).
IL-5 is essential for eosinophil differentiation (29, 30) and plays an
important role in functioning of mature eosinophils and basophils
(31, 32, 33, 34, 35). The IL-5R is composed of a unique Here we report the cloning and characterization of a STAT3 variant
which we isolated from the eosinophil cDNA library. This protein
(STAT3 MATERIALS AND METHODS Human eosinophils
were isolated from two hyper-eosinophilic individuals according to a
slight modification (41) of the method described previously (42). The
purity of the eosinophils collected was at least 95% as determined by
histochemical staining of cytospins with May-Grunwald-Giemsa stain.
Viability determined by trypan blue exclusion was more than 98%. Total
RNA was extracted from 1 × 108 eosinophils using an Rnaid
kit according to the manufacturers instructions (BIO 101 Inc.).
Poly(A)+ mRNA was extracted by oligo(dT) affinity
purification using Dynabeads Oligo(dT)25 (Dynal, Norway).
This mRNA was used to construct an EcoRI/XhoI
directional cDNA library in the Monkey COS-1 cells
were cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% heat inactivated fetal calf
serum. Human IL-5 (hIL-5) was a kind gift of Dr. D. Fattah (Glaxo
Wellcome, Stevenage). The anti-phosphotyrosine monoclonal antibody
4G10 was obtained from UBI (Lake Placid, NY). The monoclonal antibody
directed against STAT1 Oligonucleotides with the following sequence were
used in this study (only the top strand is shown): the human ICAM-1
pIRE, 5 For transfection experiments, COS
cells were split 1:3 in 6-well plates (Costar), and 2 h later the cells
were transfected with 10-20 µg of supercoiled plasmid DNA by the
calcium phosphate coprecipitation technique (46). Following 16-20 h
exposure to the calcium-phosphate precipitate, medium was refreshed,
and cells were incubated for 16 h with IL-5. Transfected cells were
subsequently harvested for luciferase assay (47) and lacZ determination
(48).
Nuclear extracts were prepared from
unstimulated and IL-5 stimulated COS cells following a previously
described procedure (49). Oligonucleotides were labeled by filling in
the cohesive ends with [ Unstimulated and
IL-5 stimulated COS cells were incubated with RIPA lysis buffer (20
mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40,
0.1% SDS, 0.5% sodium deoxycholate, 5 mM EDTA,
Na3VO4, 10 mg/ml aprotinine, 1 mM
phenylmethylsulfonyl fluoride, 1 mM leupeptin) for 15 min
on ice. The lysate was centrifuged to remove DNA and cellular debris.
The cell lysates were incubated with the anti-STAT3 polyclonal antibody
for 1 h at 4 °C. Immune complexes were then precipitated with
protein A-Sepharose for 1 h at 4 °C, washed three times with lysis
buffer, and boiled in 1 × Laemmli's sample buffer. The proteins were
electrophoresed on a SDS-polyacrylamide gel and transferred to
nitrocellulose membrane. After blocking in TBST (150 mM
NaCl, 10 mM Tris, pH 8.0, 0.3% Tween 20) with 5% bovine
serum albumin, the membrane was either incubated with the
anti-phosphotyrosine (4G10) monoclonal antibody, or with the polyclonal
anti-STAT3 antibody. After washing three times with TBST the membrane
was incubated for 1 h with peroxidase-conjugated rabbit anti-mouse or
swine anti-rabbit antibodies, respectively. In both cases the membrane
was washed five times with TBST and immunoprecipitated proteins were
visualized with enhanced chemiluminescence (ECL, Amersham). Between the
incubation with 4G10 and STAT3 antibodies the membrane was stripped
with 1% SDS, 30 mM Tris, pH 8.0, 50 mM
RESULTS In order to identify new
STAT proteins expressed in eosinophils that may play a role in IL-5
signaling, an eosinophil cDNA library was screened with a labeled
probe containing the full-length STAT cDNAs of hSTAT1, mSTAT3,
hSTAT4, and hSTAT6. After low stringency screening of the cDNA
library, several positive phage clones were isolated. The inserts of
these clones were characterized by Southern hybridization and
sequencing. All the isolated clones encode known STAT proteins
including hSTAT3, hSTAT4, and hSTAT6. However, one of the STAT3
positive cDNA clones showed a different restriction pattern
compared to the wild type STAT3 cDNA. Sequencing of this clone
revealed that it is almost identical to STAT3 but lacks an internal
part of 50 base pairs, covering nucleotides 2145-2195 near the COOH
terminus (Fig. 1A). This deletion removes
codons 716 to 732 and in addition causes a shift in the open reading
frame resulting in the formation of a stop codon after 7 amino acids
(Fig. 1B). This truncated STAT3 mRNA encodes a protein
consisting of the first 715 amino acids of STAT3 plus an additional 7
unique amino acids. Comparison of the amino acids in the
carboxyl-terminal region shows that this truncated STAT3 protein
contains the tyrosine phosphorylation site at position 704, but lacks
the conserved PMSP sequence which is a substrate for a serine kinase
(50). Based on published work (7) identifying a splice variant of STAT1
named STAT1
To investigate
the existence of the STAT3
We have
previously shown that STAT3 becomes tyrosine phosphorylated after IL-5
treatment in both BaF3 and COS cells (40). Since STAT3 Tyrosine phosphorylation of STAT proteins leads to dimerization,
translocation to the nucleus, and binding to specific binding sites on
the DNA. We therefore tested the ability of STAT3
To
compare the transcriptional activity of STAT3 with STAT3
Since all STAT proteins form homo- or heterodimers after
phosphorylation on tyrosine, a mechanism for the inhibition could be
that STAT3 and STAT3
DISCUSSION STAT proteins are a rapidly expanding family of transcription
factors that transduce short-term cytoplasmic signals elicited by
polypeptide growth factors and cytokines into long-term changes in gene
expression (52). Here, the cloning and characterization of a novel
isoform of the STAT3 transcription factor is reported, which is named
STAT3 Differential splicing in the STAT family is not unprecedented.
Schindler et al. (7) have shown that the STAT1 gene encodes
at least two different proteins, STAT1 It was previously suggested that besides tyrosine phosphorylation,
serine phosphorylation might play a crucial role in gene regulation by
STAT proteins. The serine/threonine kinase inhibitor H7 was shown to be
able to block transcriptional regulation by STAT1 and STAT3 (19, 20).
Similarly, Boulton et al. (22) reported an H-7 sensitive
phosphorylation of STAT3, but not STAT1. In addition, it was shown that
serine phosphorylation might be necessary for DNA binding by STAT3
homodimers, but not by STAT1 homo- or STAT1/STAT3 heterodimers (21). In
this paper we have shown that STAT3 The observation that STAT3 The observed dominant negative effect of STAT3 FOOTNOTES The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U30709[GenBank]. Acknowledgments We thank James E. Darnell, Jr., and Jan
Tavenier for the kind gift of plasmids. We further thank Dilnya Fattah
for hIL-5 and Paul Coffer for critically reading the manuscript.
REFERENCES
Volume 271, Number 22,
Issue of May 31, 1996
pp. 13221-13227
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
, a Splice Variant of Transcription Factor STAT3, Is a
Dominant Negative Regulator of Transcription*
,,
Glaxo Wellcome
Medicines Research Centre, Gunnels Wood Road, Stevenage,
Hertfordshire SG1 2NY, United Kingdom
) which was isolated by screening an
eosinophil cDNA library. Compared to wild-type STAT3, STAT3
lacks an internal domain of 50 base pairs located near the C terminus.
This splice product is a naturally occurring isoform of STAT3 and
encodes a 80-kDa protein. We found by reconstitution of the human IL-5R
in COS cells that like STAT3, STAT3 is phosphorylated on tyrosine
and binds to the pIRE from the ICAM-1 promoter after IL-5 stimulation.
However, STAT3 fails to activate a pIRE containing promoter in
transient transfection assays. Instead, co-expression of STAT3
inhibits the transactivation potential of STAT3. These results suggests
that STAT3 functions as a negative regulator of transcription.
(IFN-) and signaling pathway.
IFN activates a latent cytoplasmic transcription factor complex
interferon-stimulated gene factor 3 (5, 6, 7). This complex consists of a
48-kDa DNA-binding component, and the tyrosine-phosphorylated
proteins STAT1, STAT1, and STAT2 (8). By contrast, only STAT1
is tyrosine phosphorylated upon stimulation of cells with IFN-
(9).
Until now, eight members of the STAT family: STAT1, STAT1 (6, 7),
STAT2 (6), STAT3 (10, 11), STAT4 (12, 13), STAT5A, STAT5B (14, 15, 16), and
STAT6 (17) have been identified and characterized. All the STATs are
widely expressed in different cell types and tissues, except for STAT4,
which is expressed predominantly in testis and in cells of
hematopoietic origin. Phosphorylation on tyrosine of the STAT proteins
is required for dimerization, DNA-binding, and the activation of
transcription (18, 8). However, STAT1, which is a splice product of
STAT1 and lacks 38 amino acids of the carboxyl terminus, is
phosphorylated on tyrosine but is transcriptionally inactive (18).
Furthermore, we and others found that H7, which is a serine/threonine
kinase inhibitor, blocked the transactivation potential of STAT1 and/or
STAT3 (19, 20, 21, 22). These results suggest that phosphorylation of serine
residues in STAT1 and STAT3 are necessary for the transcriptional
activity of these proteins.
subunit associated with
a c subunit that is identical to those of the receptors for IL-3 and
GM-CSF (36). We and others have shown that JAK2 is activated by IL-3,
IL-5, and GM-CSF (37, 38), and constitutively associates with the
membrane-proximal region of the c subunit (39). Recently, we have
described that STAT3 activity increases in response to interleukin-5
(IL-5) in both BaF3 and COS cells ectopically expressing the hIL-5
receptor (IL-5R) (40). Although STAT3 is tyrosine phosphorylated and
activated by IL-5 in these cells, it was only activated to a very low
extent in mature eosinophils. Based on this and the observation that
multiple STATs are activated by IL-5 in eosinophils (37), we screened
an eosinophil cDNA library to identify IL-5 induced novel STAT
cDNAs.
) is a truncated form of STAT3 which is probably generated by
differential splicing. STAT3 and STAT3 protein are co-expressed in
various cell types. We found that STAT3 is rapidly phosphorylated on
tyrosine upon IL-5 stimulation of COS cells. However, although STAT3
efficiently binds to the palindromic IL-6/IFN response element
(pIRE) from the intercellular adhesion molecule 1 (ICAM-1) promoter, it
is unable to activate a promoter containing this pIRE element. We also
demonstrate that STAT3 is a strong dominant inhibitor of
transcription.
ZAPII vector as described by
the manufacturer (Stratagene). The library contained greater than 2 ×
106 primary recombinants with a background of less than
10%. The average insert size was approximately 1.5 kilobase and the
largest inserts were estimated to be greater than 5 kilobases.
/ was purchased from Transduction
Laboratories (Lexington, KY). The STAT3 rabbit polyclonal antibodies
K15 (directed against amino acids 626-640) and C20 (directed against
amino acids 750-769) were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA).
-AGCTTAGGTTTCCGGGAAAGCAC-3. The 2xpIREtkluc and pICAM1-339
reporter constructs has been described by Caldenhoven et al.
(43) and the pSV-lacZ expression vector by Shen et al. (44).
pSGhIL5R was constructed by inserting the cDNA for the human
IL-5 receptor from pBKhIL5R (45) into the
Not/KpnI sites of pSG513. pSGhIL5R was
constructed by inserting the cDNA for the human c subunit from
pSV532 (36) into the EcoRI sites of pSG513. The expression
vectors containing the cDNAs for hSTAT1, mSTAT3, and hSTAT4 were
provided by Dr. James E. Darnell, Jr. (7, 11). The hSTAT6 cDNA was
provided by Dr. Steven L. McKnight (17). Full-length STAT cDNAs
were used for the screening of the cDNA library. The hSTAT3 and
hSTAT3 were isolated from the eosinophil cDNA library and cloned
into the EcoRI site of PSG513.
-32P]dCTP using Klenow
fragment of DNA polymerase I. Gel retardation assays were carried out
according to published procedures with slight modifications (5).
Briefly, nuclear extracts (10 µg) were incubated in a final volume of
20 µl, containing 10 mM HEPES, pH 7.8, 50 mM
KCl, 1 mM EDTA, 5 mM MgCl2, 10%
(v/v) glycerol, 5 mM dithiothreitol, 2 µg of poly(dI-dC)
(Pharmacia), 20 µg of bovine serum albumin, and 1.0 ng of
32P-labeled ICAM1-pIRE oligonucleotide for 20 min at room
temperature. In competition experiments, extracts were incubated for 5
min with the indicated molar excess of unlabeled oligonucleotide prior
to the addition of labeled oligonucleotide. Supershift analysis were
performed by preincubating 10 µg of nuclear extract with 1 µl (1
µg) of anti-STAT3 antibody for 30 min on ice prior to addition of the
binding buffer and 32P-labeled probe.
-mercaptoethanol for 2 × 15 min at 55 °C.
, it is likely that the truncated form of STAT3 results
from an alternative splicing event as well. We therefore designated the
STAT3 clone with this internal deletion as STAT3. To determine
whether this isoform is not a cloning artifact we performed a
polymerase chain reaction experiment with internal primers depicted in
Fig. 1A on mRNA from the cell types mentioned below. As
expected, two polymerase chain reaction products were formed a 300-base
pair fragment and a minor 250-base pair fragment (data not shown).
Sequencing revealed that these DNA fragments corresponds to the
internal regions of STAT3 (300 base pairs) and STAT3 (250 base
pairs). Additional evidence that this isoform is not a cloning artifact
comes from the independent isolation of this cDNA by Schaefer
et al. (51), who isolated this cDNA via a two hybrid
screen with a N-terminal segment of c-Jun.
Fig. 1.
Nucleotide and amino acid sequence of the
COOH-terminal regions of human STAT3 and STAT3. A, STAT3
and STAT3 cDNAs were isolated from a human eosinophil cDNA
library, subcloned, and sequenced. As can be observed, STAT3
contains an internal deletion of 50 nucleotides. The positions of the
stop codons used in STAT3 and STAT3 and the primers used for the
detection of STAT3 and STAT3 mRNA are indicated. B,
schematic representation of the amino acid sequences of STAT3 and
STAT3 around the internal deletion. Positions of the Src homology 2
(SH2), SH3, and tyrosine phosphorylation (Tyr704) domains
are indicated. Due to the deletion in STAT3, the reading frame is
switched and a stop codon is generated 7 amino acids downstream of the
internal deletion.
Is Expressed in Different Cell Types
protein and to establish its tissue
distribution, we performed a STAT3 immunoprecipitation from different
cell types. For this purpose, cell lysates were prepared from U937,
HL-60, BaF3 cells, eosinophils, COS cells, and COS cells transfected
with the cDNA encoding for STAT3 (COS/STAT3). The proteins
were precipitated with a specific STAT3 antibody directed against
residues 626-640 and immunoblotted with this STAT3 antibody. Fig.
2A shows that untransfected COS cells express
only a low amount of the 89-kDa STAT3 protein. However, overexpressing
STAT3 in COS cells results in the appearance of an 80-kDa protein
which comigrated with an endogenously expressed 80-kDa protein in the
other cell types analyzed with the STAT3 antibody. The expression of
this 80-kDa protein varies between the different cell types studied
(Fig. 2A). This 80-kDa protein was also found in blood
monocytes, lymphocytes, and neutrophils (data not shown). To ascertain
that the endogenously expressed 80-kDa protein was STAT3, we
precipitated the proteins with a STAT3 antibody directed against amino
acids 750-769, a domain lacking in the STAT3 protein. Using this
antibody we detect only the 89-kDa wild type STAT3 protein in
COS/STAT3, COS/STAT3, U937, and HL-60 cells (Fig. 2C, lanes
5-8). When proteins from these cells were precipitated with STAT3
antibodies directed against residues 629-640, two STAT3 proteins
appeared of 89 and 80 kDa, except for the COS/STAT3 cells which shows
only the wild type STAT3 (Fig. 2C, lanes 1-4). We therefore
conclude that the endogenously expressed 80-kDa protein in primary
cells as well as in the cell lines is likely to be STAT3 since it is
not recognized by an antibody against the N terminus which is lacking
from STAT3.
Fig. 2.
Expression and tyrosine phosphorylation of
the STAT3 protein. A, whole cell extracts from COS
(lane 1), COS transfected with STAT3 (lane 2),
BaF3 (lane 3), HL-60 (lane 4), U937 (lane
5), and eosinophils (lane 6) were monitored for the
presence of STAT3 and STAT3 by immunoprecipitation with a STAT3
specific antibody (amino acids 629-640) followed by Western blotting
with the same antibody. The protein of about 89 kDa represents STAT3,
while the smaller protein (80 kDa) is STAT3. STAT3 and STAT3 are
co-expressed in different cell types, although at different ratios.
B, COS cells were transfected with the IL-5R and
cDNAs together with the pSG5 expression vector (lanes 1
and 2), STAT3 (lanes 3 and 4), or
STAT3 (lanes 5 and 6). 48 h after
transfection, cells received IL-5 for 15 min (lanes 2, 4,
and 6), after which cells were lyzed and STAT3 was
immunoprecipitated. The blot was first probed with an
anti-phosphotyrosine antibody (upper panel), stripped, and
probed with the STAT3 antibody (lower panel). IL-5 causes a
strong increase in tyrosine phosphorylation of both STAT3 and STAT3.
The faster migrating bands observed in lanes 3 and
4 (lower panel) are sometimes observed, and are
probably the result of degradation of STAT3. C, whole cell
extracts from COS cells transfected with STAT3 (lanes 1 and
5), or STAT3b (lanes 2 and 6), U937
(lane 3 and 7), and HL-60 cells (lanes
4 and 8) were immunoprecipitated with a STAT3 antibody
(629-640) (lanes 1-4) or a STAT3 antibody (amino acids
750-769) (lanes 5-8) and blotted with STAT3 antibody
(629-640). The endogenously expressed 80-kDa protein is likely to be
STAT3.
still retains
the tyrosine residue necessary for STAT3 activation, we were interested
whether STAT3 is phosphorylated on tyrosine after IL-5 stimulation.
To test this prediction we co-transfected COS cells with expression
vectors encoding both subunits of the IL-5 receptor (IL-5R and
IL-5R), together with STAT3 or STAT3. We immunoprecipitated STAT3
from unstimulated and IL-5 stimulated COS cells and tyrosine
phosphorylation was then monitored by Western blotting using the
anti-phosphotyrosine antibody 4G10. Fig. 2B shows that both
wild-type STAT3 and STAT3 are phosphorylated on tyrosine after IL-5
stimulation. However, although the expression of STAT3 protein is
less then wild-type STAT3, the amount of tyrosine phosphorylation is
higher. In addition, even in the absence of IL-5 signaling, we detected
some basal level tyrosine phosphorylation of STAT3, which we have
never observed with STAT3.
to bind DNA and
compared this with wild-type STAT3. COS cells expressing the IL-5R and
either wild-type STAT3 or STAT3 were treated with IL-5 for 15 min
and nuclear extracts were prepared. When these nuclear extracts were
assayed in a gel retardation assay for binding to a
32P-labeled ICAM-1 pIRE, an increase in STAT3 and STAT3
binding was observed after IL-5 treatment (Fig. 3).
Unexpectedly, however, the STAT3 complex migrated faster than the
STAT3 complex. The STAT3 complex is specific because an
anti-STAT3 antibody produced a supershift, while STAT1 antiserum had no
effect on this complex. Furthermore, the DNA binding activity of
STAT3 is higher than STAT3, which is probably due to the higher
amount of tyrosine-phosphorylated STAT3 proteins observed (Fig.
2B). In addition, we observed some basal level DNA binding
activity by STAT3 in unstimulated cells, which is in agreement with
the observed tyrosine phosphorylation in unstimulated cells (Fig.
2B). We can conclude that both wild-type STAT3 and STAT3
are tyrosine phosphorylated after IL-5 stimulation which results in an
increase in DNA binding of both proteins.
Fig. 3.
Both STAT3 and STAT3 bind to DNA after
activation by IL-5. The hIL-5 receptor and STAT3 or STAT3 were
expressed in COS cells. These cells were either untreated or treated
for 30 min with IL-5, after which nuclear extracts were prepared.
Nuclear extract were assayed for binding to the 32P-labeled
ICAM-1 pIRE in band shift experiments. For competition experiments,
extracts were preincubated for 5 min with a 50-fold molar excess of
unlabeled oligonucleotide as indicated. For supershift analysis, the
extracts were incubated with either anti-STAT1 or anti-STAT3
antibodies for 30 min before the addition of the
32P-labeled ICAM-1 pIRE. IL-5 clearly induces binding of
STAT3 and STAT3 to the ICAM-1 pIRE.
Acts as a Dominant Transcriptional Repressor
, we
transiently transfected COS cells with expression vectors for the
IL-5R, wild-type STAT3, or STAT3 together with a luciferase reporter
construct, containing two copies of the pIRE from the ICAM-1 promoter.
Transfection of the wild-type STAT3 cDNA shows an IL-5 dependent
15-fold increase in luciferase activity (Fig.
4A). By contrast, transfection of STAT3
gave almost no increase in luciferase activity after IL-5 stimulation.
An explanation for this could be that the region which is absent from
STAT3 contains a domain important for transactivation. We further
determined the effect of STAT3 on transactivation mediated by STAT3.
We tested this by co-transfection of an increasing amount of STAT3
expression vector together with a constant amount of STAT3 expression
vector. The total amount of DNA was kept constant by adding the pSG5
vector. In this experiment we see that transactivation by STAT3 is
already inhibited by low amounts of STAT3 (Fig. 4B).
Western blotting indicated that STAT3 expression did not alter the
level of STAT3 (data not shown). These results implicate that STAT3
can act as a dominant negative regulator of STAT3-mediated
transcription. To make sure that this dominant effect of STAT3 also
occurs on a natural promoter we used the ICAM-1 promoter containing the
IRE in transient transfections experiments. COS cells were transfected
with this reporter construct together with STAT3, STAT3, or a
combination of both STAT proteins and stimulated with IL-5. We found
that although STAT3 is an activator of the ICAM-I promoter, STAT3 is
transcriptionally inactive on the ICAM-1 promoter and acts as a
dominant negative regulator of STAT3-mediated transcription. (Fig.
4C).
Fig. 4.
STAT3 is a dominant negative regulator of
transcription. A, COS cells were transfected with the IL-5R,
a pIRE containing luciferase reporter construct and STAT3 or STAT3.
24 h post-transfection, cells were stimulated for 16 h with IL-5
(10
10 M), after which transcriptional
activation was measured by assaying for luciferase activity. Fold
induction represents luciferase activity in IL-5-treated cells compared
to untreated cells, and is the mean of three independent experiments.
STAT3 is unable to support IL-5 induced activation of the pIRE
reporter construct. B, COS cells were transfected as
described in A with different amounts of STAT3 and STAT3
as indicated. Low amounts of STAT3 already significantly decrease
trans-activation by STAT3, while high amounts of STAT3 completely
inhibit STAT3-mediated transcriptional activation. C, COS
cells were transfected as described in A and B.
We used the ICAM-1 promoter as a luciferase reporter construct
(pIC-339luc). STAT3 is also transcriptionally inactive on the
natural ICAM-1 promoter.
form heterodimers which are unable or less able
to mediate transactivation. To investigate this, we prepared nuclear
extracts from IL-5-treated COS cells transfected with STAT3 and
increasing amounts of STAT3. We assayed these nuclear extracts for
binding to the ICAM-1 pIRE and performed a long run gel retardation to
resolve the different complexes. As we already observed, the affinity
of STAT3 homodimers (Fig. 5A, lane 6) to
the pIRE is higher then the binding of STAT3 homodimers (lane
1). Interestingly, an intermediate complex C2 was observed when
STAT3 is co-transfected together with STAT3, which is likely to
consist of a STAT3/STAT3 heterodimer. To resolve all three
DNA-binding complexes, lanes 2-7 were exposed less than
lane 1. We also identified the components in these complexes
using two specific STAT3 antibodies, one directed against residues
629-640 supershifting both STAT3 and STAT3 (Fig. 5B, lanes
2 and 5), the other against residues 750-769 which
recognized only STAT3 (lanes 3 and 6). We further
show that the STAT3 (750-769) antibody which recognizes only STAT3
supershifted only the STAT3/STAT3 heterodimer and had no effect on
the STAT3 homodimer (lane 9). These results clearly
demonstrate that STAT3 and STAT3 form heterodimeric DNA-binding
complexes.
Fig. 5.
Heterodimerization between STAT3 and
STAT3. A, COS cells were transfected with the IL-5R and
different amounts of STAT3 and STAT3. 48 h after transfection, cells
were treated with IL-5 for 15 min and DNA binding activity was
monitored using a 32P-labeled pIRE. Co-expression of STAT3
(C3) and STAT3 (C1) results in the formation
of a heterodimeric complex (C2). B, nuclear
extracts from COS cells transfected with STAT3, STAT3, or
STAT3/STAT3, stimulated for 15 min with IL-5 were preincubated with
two different STAT3 antibodies STAT3 Ab (629-640) and STAT3 Ab
(750-769). DNA binding activity was monitored using a
32P-pIRE. These data clearly show the formation of STAT3
homodimers and STAT3/STAT3 heterodimers.
in analogy with STAT1/STAT1. Although STAT3
is
phosphorylated on tyrosine upon IL-5 stimulation and binds efficiently
to the pIRE from the ICAM-1 promoter, it fails to support pIRE-driven
transcription in IL-5-stimulated cells. Moreover, STAT3 is an
efficient dominant negative regulator of STAT3-mediated
transcription.
and STAT1, that are
generated by alternative splicing. Like STAT3, STAT1 can be
phosphorylated on tyrosine but fails to activate transcription (18).
However, whether STAT1 can act as a dominant negative regulator of
STAT1 was never investigated. The splicing event occurs at a highly
homologous position in STAT1 and STAT3 (22, 52), suggesting that STAT1
and STAT3 might have a conserved exonic organization as was previously
demonstrated for STAT1 and STAT2 (53). Verification of this hypothesis,
however, awaits deciphering of the precise exonic structure of the
STAT3 gene.
is efficiently phosphorylated on
tyrosine upon IL-5 stimulation (Fig. 2B), which leads to a
strong increase in DNA binding by STAT3 (Fig. 3). However, in
contrast to STAT3, STAT3 is unable to mediate transactivation via a
pIRE containing promoter. Examination of the amino acids deleted from
STAT3 shows the presence of a large number of serine and threonine
residues, of which serine 727 is conserved between STAT1, STAT3,
STAT4, and STAT5, but not STAT1. Interestingly, this serine lies
within a highly conserved PMSP sequence that was previously shown to be
a microtubule-associated protein kinase recognition sequence (50). The
importance of this serine was very recently shown by Wen et
al. (20), who showed that this serine is inducibly phosphorylated
in both STAT1 and STAT3. Furthermore, when they mutated this serine
to alanine (STAT1S), trans-activation was decreased 5-fold, although
STAT1S was still able to support an IFN-induced 5-fold increase
in transcription, whereas STAT1 is completely inactive in this
system (20). Similarly, mutation of serine 727 in STAT3 also decreased
transactivation about 2.5-fold, although the mutant was still able to
support an 8-fold induction of INF activation site-mediated
transcription (20). The lack of serine 727 in STAT3 explains some,
but not all of the results presented in this paper. STAT3 is
completely unable to mediate transcriptional activation in COS cells
(Fig. 4A), whereas STAT3 with the 727 mutation is still a
relatively good trans-activator in U3A cells (20). This might be caused
by cell type-specific differences between COS and U3A. Alternatively,
there might be more phosphorylated residues present in the region that
is deleted from STAT3 which contribute to transcriptional
activation. Finally, the basal level tyrosine phosphorylation (Fig.
2B) and DNA binding (Fig. 3) observed with STAT3 in
unstimulated COS cells represents a striking difference with both
STAT3-727 and STAT1. Although we do not have any experimental data
to support this hypothesis, this observation suggests the presence of a
domain in the COOH-terminal 55 amino acids of STAT3 that is somehow
able to block STAT3 phosphorylation by JAK kinases in unstimulated
cells.
is transcriptionally inactive is in
contrast with data published recently by Schaefer et al.
(51). They have identified STAT3 via a two-hybrid system as a
protein capable of binding to the NH2-terminal part of the
c-Jun protein. Furthermore, they have shown that STAT3 is
transcriptionally active on the 2-macroglobulin promoter
in the absence of added cytokines. This constitutive transactivation
potential is consistent with our own results showing a constitutive
tyrosine phosphorylation of STAT3. The opposite effects found on the
transactivation potential of STAT3 can be due to the promoter
targets used in both studies. We have used an artificial reporter
containing only STAT binding sites and the natural ICAM-I promoter,
while the 2-macroglobulin promoter used by Schaefer
et al. (51) contains a STAT binding site closely linked to a
Jun binding site. Occupation of the Jun binding site by members of the
Jun/Fos family might somehow alter the transcription activation
potential of STAT3. Opposite effects by a single transcription
factor on different promoters has also been described for hormone
receptors which can either activate or repress gene transcription
depending on the promoter (54). Further experiments are required for
deciphering the different roles that STAT3 might have in
transcriptional regulation.
over STAT3 might be
caused by two different mechanisms. One possibility might be that
STAT3 homodimers have a higher affinity for the pIRE, and therefore
occupy these sites on the DNA even when STAT3 is more abundantly
expressed. Indeed, we have observed that while STAT3 expression was
2-fold lower in transfected COS cells (Fig. 2B), DNA binding
by STAT3 was 2-3-fold higher compared to STAT3 (Fig. 3). This is
likely to be caused by the higher extent of tyrosine phosphorylation
observed in STAT3 (Fig. 2B). On the other hand, since
dimerization of STAT proteins is required for the formation of
transcriptionally active DNA binding complexes (52), heterodimerization
between STAT3 and STAT3 might be involved in the dominant negative
effect. In Fig. 5 we show that STAT3 and STAT3 indeed form a
heterodimer. Moreover, even a small amount of STAT3 is sufficient to
disrupt all the STAT3 homodimers and drive them into STAT3/STAT3
heterodimers. However, at this ratio between STAT3 and STAT3 (6:2
µg), we still observe transcriptional activation of a pIRE containing
plasmid (Fig. 4B), although it is 50% less compared to
STAT3 alone. This suggests that the STAT3/STAT3 heterodimer is able
to support IL-5 induced transcription, albeit with a lower efficiency
than the STAT3 homodimer. Taken together, the observed dominant
negative effect of STAT3 is likely to be caused by a combination of
transcriptionally inactive STAT3 homodimers with high DNA binding
activity and STAT3/STAT3 heterodimers which are weak transcriptional
activators. The generation of transcriptional activators and repressors
from the same gene is not unprecedented (55). mTFE3, a murine
transcription factor involved in the activation of immunoglobulin heavy
chain transcription, is turned into a repressor by a splicing event
that removes part of the activation domain (56). Similarly, a splicing
event removing part of the activation domain of FosB results in the
expression of 
FosB, an inhibitor of Fos/Jun transcriptional activity
(57), while splicing out the activation domains of CREM
generates
CREM, which, despite the fact that it retains the protein kinase A
phosphoacceptor site, is an efficient antagonist of cAMP-induced
transcription (58, 59). The use of alternative initiation codons can
also be a mechanism to generate activators and repressors from the same
mRNA, as was reported for liver-enriched activator protein and
liver-enriched inhibitory protein (60) and for CREM and S-CREM (61). In
this paper we report that an alternative splicing event removing 55
amino acids from the COOH terminus of STAT3 generates STAT3, an
efficient repressor of STAT3-mediated transcription. Although it is
likely that the removal of serine 727 contributes to this switch from
activator to repressor, the precise molecular mechanism awaits further
mutational analysis of STAT3, since the nature of the activation domain
of STAT3 remains to be elucidated. As was described previously for
mTFE3, FosB, CREM, and liver-enriched activator protein (55), we found
that the activator STAT3 and the repressor STAT3 are co-expressed in
a wide variety of cell types (Fig. 2A), although the ratio
between the two differs between cell types studied. Cell type-specific
differences in the ratio between STAT3 and STAT3 could lead to
differences in the response to IL-5 or other activators of STAT3. It
would therefore be interesting to determine the mechanism by which the
ratio between STAT3 and STAT3 is modulated in a cell type-specific
manner. Another challenge will be to find physiological processes in
which the ratio between STAT3 and STAT3 is altered in a temporal or
spatial fashion due to signal transduction dependent alternative
splicing.
§
To whom correspondence should be addressed: Dept. of Pulmonary
Diseases, G03.550, University Hospital Utrecht, Heidelberglaan 100,
3584 CX Utrecht, The Netherlands. Tel.: 31-30-507134; Fax:
31-30-542155.
1
The abbreviations used are: STAT, signal
transducer and activator of transcription; JAK, Janus kinase; IFN,
interferon; IL-5, interleukin-5; IL-5R, interleukin-5 receptor; GM-CSF,
granulocyte macrophage-colony stimulating factor; IRE,
interferon/IL-6-responsive element; pIRE, palindromic IL-6/IFN
response element; ICAM-1, intercellular adhesion molecule-1; CREM,
cAMP-responsive element modulator; S-CREM, short CREM.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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G. Niu, K. H. Shain, M. Huang, R. Ravi, A. Bedi, W. S. Dalton, R. Jove, and H. Yu Overexpression of a Dominant-Negative Signal Transducer and Activator of Transcription 3 Variant in Tumor Cells Leads to Production of SolubleFactors That Induce Apoptosis and Cell Cycle Arrest Cancer Res., April 1, 2001; 61(8): 3276 - 3280. [Abstract] [Full Text]
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H. Chen, J. M. Lee, Y. Zong, M. Borowitz, M. H. Ng, R. F. Ambinder, and S. D. Hayward Linkage between STAT Regulation and Epstein-Barr Virus Gene Expression in Tumors J. Virol., March 15, 2001; 75(6): 2929 - 2937. [Abstract] [Full Text]
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C. T. Sherman and A. R. Brasier Role of Signal Transducers and Activators of Transcription 1 and -3 in Inducible Regulation of the Human Angiotensinogen Gene by Interleukin-6 Mol. Endocrinol., March 1, 2001; 15(3): 441 - 457. [Abstract] [Full Text]
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Z. Xia, R. R. Salzler, D. P. Kunz, M. R. Baer, L. Kazim, H. Baumann, and M. Wetzler A Novel Serine-dependent Proteolytic Activity Is Responsible for Truncated Signal Transducer and Activator of Transcription Proteins in Acute Myeloid Leukemia Blasts Cancer Res., February 1, 2001; 61(4): 1747 - 1753. [Abstract] [Full Text]
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M.-L. Hakansson-Ovesjo, M. Collin, and B. Meister Down-Regulated STAT3 Messenger Ribonucleic Acid and STAT3 Protein in the Hypothalamic Arcuate Nucleus of the Obese Leptin-Deficient (ob/ob) Mouse Endocrinology, November 1, 2000; 141(11): 3946 - 3955. [Abstract] [Full Text] [PDF]
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B. Emanuelli, P. Peraldi, C. Filloux, D. Sawka-Verhelle, D. Hilton, and E. Van Obberghen SOCS-3 Is an Insulin-induced Negative Regulator of Insulin Signaling J. Biol. Chem., May 19, 2000; 275(21): 15985 - 15991. [Abstract] [Full Text] [PDF]
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 Y.-T. Tsai, Y.-H. Su, S.-S. Fang, T.-N. Huang, Y. Qiu, Y.-S. Jou, H.-m. Shih, H.-J. Kung, and R.-H. Chen Etk, a Btk Family Tyrosine Kinase, Mediates Cellular Transformation by Linking Src to STAT3 Activation Mol. Cell. Biol., March 15, 2000; 20(6): 2043 - 2054. [Abstract] [Full Text]
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A. C. Ward, I. Touw, and A. Yoshimura The Jak-Stat pathway in normal and perturbed hematopoiesis Blood, January 1, 2000; 95(1): 19 - 29. [Full Text] [PDF]
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J. Turkson, T. Bowman, J. Adnane, Y. Zhang, J. Y. Djeu, M. Sekharam, D. A. Frank, L. B. Holzman, J. Wu, S. Sebti, et al. Requirement for Ras/Rac1-Mediated p38 and c-Jun N-Terminal Kinase Signaling in Stat3 Transcriptional Activity Induced by the Src Oncoprotein Mol. Cell. Biol., November 1, 1999; 19(11): 7519 - 7528. [Abstract] [Full Text] [PDF]
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G. Niu, R. Heller, R. Catlett-Falcone, D. Coppola, M. Jaroszeski, W. Dalton, R. Jove, and H. Yu Gene Therapy with Dominant-negative Stat3 Suppresses Growth of the Murine Melanoma B16 Tumor in Vivo Cancer Res., October 1, 1999; 59(20): 5059 - 5063. [Abstract] [Full Text] [PDF]
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A. Woetmann, M. Nielsen, S. T. Christensen, J. Brockdorff, K. Kaltoft, A.-M. Engel, S. Skov, C. Brender, C. Geisler, A. Svejgaard, et al. Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3 PNAS, September 14, 1999; 96(19): 10620 - 10625. [Abstract] [Full Text] [PDF]
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R. P. de Groot, J. A.M. Raaijmakers, J.-W. J. Lammers, R. Jove, and L. Koenderman STAT5 Activation by BCR-Abl Contributes to Transformation of K562 Leukemia Cells Blood, August 1, 1999; 94(3): 1108 - 1112. [Abstract] [Full Text] [PDF]
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M. A. Sherman, V. H. Secor, and M. A. Brown IL-4 Preferentially Activates a Novel STAT6 Isoform in Mast Cells J. Immunol., March 1, 1999; 162(5): 2703 - 2708. [Abstract] [Full Text] [PDF]
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S. L. Wyszomierski, J. Yeh, and J. M. Rosen Glucocorticoid Receptor/Signal Transducer and Activator of Transcription 5 (STAT5) Interactions Enhance STAT5 Activation by Prolonging STAT5 DNA Binding and Tyrosine Phosphorylation Mol. Endocrinol., February 1, 1999; 13(2): 330 - 343. [Abstract] [Full Text]
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A. Chakraborty, K. F. Dyer, M. Cascio, T. A. Mietzner, and D. J. Tweardy Identification of a Novel Stat3 Recruitment and Activation Motif Within the Granulocyte Colony-Stimulating Factor Receptor Blood, January 1, 1999; 93(1): 15 - 24. [Abstract] [Full Text] [PDF]
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 A. R. Simon, U. Rai, B. L. Fanburg, and B. H. Cochran Activation of the JAK-STAT pathway by reactive oxygen species Am J Physiol Cell Physiol, December 1, 1998; 275(6): C1640 - C1652. [Abstract] [Full Text] [PDF]
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T. B. van Dijk, B. Baltus, E. Caldenhoven, H. Handa, J. A.M. Raaijmakers, J.-W. J. Lammers, L. Koenderman, and R. P. de Groot Cloning and Characterization of the Human Interleukin-3 (IL-3)/IL-5/ Granulocyte-Macrophage Colony-Stimulating Factor Receptor beta c Gene: Regulation by Ets Family Members Blood, November 15, 1998; 92(10): 3636 - 3646. [Abstract] [Full Text] [PDF]
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E. Caldenhoven, T. B. van Dijk, A. Tijmensen, J. A.M. Raaijmakers, J.-W. J. Lammers, L. Koenderman, and R. P. de Groot Differential Activation of Functionally Distinct STAT5 Proteins by IL-5 and GM-CSF During Eosinophil and Neutrophil Differentiation from Human CD34+ Hematopoietic Stem Cells Stem Cells, November 1, 1998; 16(6): 397 - 403. [Abstract] [Full Text]
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R. Imamura, E. S. Masuda, Y. Naito, S.-i. Imai, T. Fujino, T. Takano, K.-i. Arai, and N. Arai Carboxyl-Terminal 15-Amino Acid Sequence of NFATx1 Is Possibly Created by Tissue-Specific Splicing and Is Essential for Transactivation Activity in T Cells J. Immunol., October 1, 1998; 161(7): 3455 - 3463. [Abstract] [Full Text] [PDF]
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M. Dajee, G. H. Fey, and J. S. Richards Stat 5b and the Orphan Nuclear Receptors Regulate Expression of the {alpha}2-Macroglobulin ({alpha}2M) Gene in Rat Ovarian Granulosa Cells Mol. Endocrinol., September 1, 1998; 12(9): 1393 - 1409. [Abstract] [Full Text]
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T. Mikita, M. Kurama, and U. Schindler Synergistic Activation of the Germline {epsilon} Promoter Mediated by Stat6 and C/EBP{beta} J. Immunol., August 15, 1998; 161(4): 1822 - 1828. [Abstract] [Full Text] [PDF]
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C. Bovolenta, L. Testolin, L. Benussi, P. M.-J. Lievens, and E. Liboi Positive Selection of Apoptosis-resistant Cells Correlates with Activation of Dominant-Negative STAT5 J. Biol. Chem., August 14, 1998; 273(33): 20779 - 20784. [Abstract] [Full Text] [PDF]
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T. Bellido, C. A. O'Brien, P. K. Roberson, and S. C. Manolagas Transcriptional Activation of the p21WAF1,CIP1,SDI1 Gene by Interleukin-6 Type Cytokines. A PREREQUISITE FOR THEIR PRO-DIFFERENTIATING AND ANTI-APOPTOTIC EFFECTS ON HUMAN OSTEOBLASTIC CELLS J. Biol. Chem., August 14, 1998; 273(33): 21137 - 21144. [Abstract] [Full Text] [PDF]
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S. Skov, M. Nielsen, S. Bregenholt, N. Odum, and M. H. Claesson Activation of Stat-3 Is Involved in the Induction of Apoptosis After Ligation of Major Histocompatibility Complex Class I Molecules on Human Jurkat T Cells Blood, May 15, 1998; 91(10): 3566 - 3573. [Abstract] [Full Text] [PDF]
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J. Turkson, T. Bowman, R. Garcia, E. Caldenhoven, R. P. De Groot, and R. Jove Stat3 Activation by Src Induces Specific Gene Regulation and Is Required for Cell Transformation Mol. Cell. Biol., May 1, 1998; 18(5): 2545 - 2552. [Abstract] [Full Text]
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K. Inoue, H. Tamaki, H. Ogawa, Y. Oka, T. Soma, T. Tatekawa, Y. Oji, A. Tsuboi, E. H. Kim, M. Kawakami, et al. Wilms' Tumor Gene (WT1) Competes With Differentiation-Inducing Signal in Hematopoietic Progenitor Cells Blood, April 15, 1998; 91(8): 2969 - 2976. [Abstract] [Full Text] [PDF]
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T. B. van Dijk, E. Caldenhoven, J. A.M. Raaijmakers, J.-W. J. Lammers, L. Koenderman, and R. P. de Groot The Role of Transcription Factor PU.I in the Activity of the Intronic Enhancer of the Eosinophil-Derived Neurotoxin (RNS2) Gene Blood, March 15, 1998; 91(6): 2126 - 2132. [Abstract] [Full Text] [PDF]
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B. K. R. Patel, J. H. Pierce, and W. J. LaRochelle Regulation of interleukin 4-mediated signaling by naturally occurring dominant negative and attenuated forms of human Stat6 PNAS, January 6, 1998; 95(1): 172 - 177. [Abstract] [Full Text] [PDF]
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S. Takemoto, J. C. Mulloy, A. Cereseto, T.-S. Migone, B. K. R. Patel, M. Matsuoka, K. Yamaguchi, K. Takatsuki, S. Kamihira, J. D. White, et al. Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins PNAS, December 9, 1997; 94(25): 13897 - 13902. [Abstract] [Full Text] [PDF]
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M. J. Walter, D. C. Look, R. M. Tidwell, W. T. Roswit, and M. J. Holtzman Targeted Inhibition of Interferon-gamma -dependent Intercellular Adhesion Molecule-1 (ICAM-1) Expression Using Dominant-Negative Stat1 J. Biol. Chem., November 7, 1997; 272(45): 28582 - 28589. [Abstract] [Full Text] [PDF]
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J. Ng and D. Cantrell STAT3 Is a Serine Kinase Target in T Lymphocytes. INTERLEUKIN 2 AND T CELL ANTIGEN RECEPTOR SIGNALS CONVERGE UPON SERINE 727 J. Biol. Chem., September 26, 1997; 272(39): 24542 - 24549. [Abstract] [Full Text] [PDF]
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M. Nielsen, K. Kaltoft, M. Nordahl, C. Ropke, C. Geisler, T. Mustelin, P. Dobson, A. Svejgaard, and N. Odum Constitutive activation of a slowly migrating isoform of Stat3 in mycosis fungoides: Tyrphostin AG490 inhibits Stat3 activation and growth of mycosis fungoides tumor cell lines PNAS, June 24, 1997; 94(13): 6764 - 6769. [Abstract] [Full Text] [PDF]
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K. K. Kuropatwinski, C. De Imus, D. Gearing, H. Baumann, and B. Mosley Influence of Subunit Combinations on Signaling by Receptors for Oncostatin M, Leukemia Inhibitory Factor, and Interleukin-6 J. Biol. Chem., June 13, 1997; 272(24): 15135 - 15144. [Abstract] [Full Text] [PDF]
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H. Kim and H. Baumann The Carboxyl-terminal Region of STAT3 Controls Gene Induction by the Mouse Haptoglobin Promoter J. Biol. Chem., June 6, 1997; 272(23): 14571 - 14579. [Abstract] [Full Text] [PDF]
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R. A. Kirken, M. G. Malabarba, J. Xu, X. Liu, W. L. Farrar, L. Hennighausen, A. C. Larner, P. M. Grimley, and H. Rui Prolactin Stimulates Serine/Tyrosine Phosphorylation and Formation of Heterocomplexes of Multiple Stat5 Isoforms in Nb2 Lymphocytes J. Biol. Chem., May 30, 1997; 272(22): 14098 - 14103. [Abstract] [Full Text] [PDF]
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R. P. de Groot, T. B. van Dijk, E. Caldenhoven, P. J. Coffer, J. A.M. Raaijmakers, J.-W. J. Lammers, and L. Koenderman Activation of 12-O-Tetradecanoylphorbol-13-acetate Response Element- and Dyad Symmetry Element-dependent Transcription by Interleukin-5 Is Mediated by Jun N-terminal Kinase/Stress-activated Protein Kinase Kinases J. Biol. Chem., January 24, 1997; 272(4): 2319 - 2325. [Abstract] [Full Text] [PDF]
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Y. Zhang, J. Turkson, C. Carter-Su, T. Smithgall, A. Levitzki, A. Kraker, J. J. Krolewski, P. Medveczky, and R. Jove Activation of Stat3 in v-Src-transformed Fibroblasts Requires Cooperation of Jak1 Kinase Activity J. Biol. Chem., August 4, 2000; 275(32): 24935 - 24944. [Abstract] [Full Text] [PDF]
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O. K. Park, L. K. Schaefer, W. Wang, and T. S. Schaefer Dimer Stability as a Determinant of Differential DNA Binding Activity of Stat3 Isoforms J. Biol. Chem., October 6, 2000; 275(41): 32244 - 32249. [Abstract] [Full Text] [PDF]
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T. Bowman, M. A. Broome, D. Sinibaldi, W. Wharton, W. J. Pledger, J. M. Sedivy, R. Irby, T. Yeatman, S. A. Courtneidge, and R. Jove Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis PNAS, June 19, 2001; 98(13): 7319 - 7324. [Abstract] [Full Text] [PDF]
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T. R. Faruqi, D. Gomez, X. R. Bustelo, D. Bar-Sagi, and N. C. Reich Rac1 mediates STAT3 activation by autocrine IL-6 PNAS, July 31, 2001; 98(16): 9014 - 9019. [Abstract] [Full Text] [PDF]
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