From the Institute of Molecular and Cell Biology, Singapore 117609, Singapore
Received for publication, August 21, 2000, and in revised form, March 14, 2001
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ABSTRACT |
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Stat3 is a latent transcription factor activated
by various cytokines and growth factors. Phosphorylation on Tyr-705 is
a prerequisite for dimer formation, nuclear translocation, binding to
its cognate DNA sequences, and regulation of the target gene transcription. Ser-727 phosphorylation of Stat3 plays an additional role in the regulation of transcription. MEK kinase 1 (MEKK1) is a
mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) that
activates the c-Jun NH2-terminal kinase signaling
pathway. Here we report that MEKK1 is involved in the regulation of
Stat3 activation by growth factors. Kinase-inactive MEKK1 inhibits
Stat3 phosphorylation on tyrosine and serine, and its transcriptional activity stimulated by epidermal growth factor and platelet-derived growth factor in different cell types. In contrast, active MEKK1 induces Stat3 tyrosine and serine phosphorylation leading to a functionally active Stat3 capable of binding DNA and enhancing transcription. Ser-727 is phosphorylated by MEKK1 in vitro,
whereas Tyr-705 phosphorylation induced by MEKK1 involves Src and Janus kinases in vivo. These data demonstrate for the first time
a novel role of MEKK1 to modulate tyrosine kinases that results in the activation of specific members of STAT family.
STAT1 family was first
identified in the regulation of interferon-inducible gene transcription
(1). These transcription factors were named Signal
Transducers and Activators of
Transcription, by virtue of their novel and unique dual
functions as signaling molecules in the cytoplasm and as transcription
factors following nuclear translocation. Concurrent to the explosion of
STAT family was the discovery of a group of tyrosine kinases called
Janus kinases (JAKs) in interferon and cytokine signaling (2, 3). Upon
cytokine stimulation, these receptor-associated kinases
transphosphorylate themselves and the receptors, creating recruitment
sites for binding proteins containing the Src homology 2 (SH2) domains,
such as STATs. The STATs are subsequently phosphorylated by JAKs on a single tyrosine site at the COOH terminus, form homo- or heterodimers via the reciprocal interactions between SH2 domain and the
phosphorylated tyrosine, and translocate into the nucleus where they
bind to DNA and regulate the transcription of target genes (reviewed in Refs. 4-6).
There are seven known mammalian STAT proteins, denoted by Stat1,
Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6, that are activated in
various cytokine signaling. Stat3 was identified as an acute-phase
response factor activated by interleukin-6 (IL-6) in mouse liver and by
homology to Stat1 (7, 8). In addition to cytokines, Stat3 is also
strongly activated by growth factors, such as EGF,
platelet-derived growth factor (PDGF), and colony-stimulating factor-1
(8-11). Although the mechanisms of Stat3 activation by growth
factors are less defined compared with cytokines, the intrinsic tyrosine kinase activity of the EGF receptor (EGF-R) and the
nonreceptor tyrosine kinases, Src and JAKs, are implicated in its
tyrosine phosphorylation and activation (12-15). IL-6 and EGF also
induce serine phosphorylation of Stat3 on Ser-727, and this event
appears to play an additional role in the regulation of Stat3-targeted gene transcription, contributing to its maximum transcriptional activity (16, 17). The MAPK family is composed of extracellular signal-regulated kinase (ERK), induced by growth factors and cytokines, JNK/stress-activated protein kinase (SAPK), and p38/HOG1 (p38), both
activated by pro-inflammatory cytokines and environmental stresses
(reviewed in Refs. 18 and 19). Phosphorylation at Ser-727 of Stat3 by
all three MAPK family members has been reported in response to
different extracellular stimuli (17, 20-24), suggesting a cross-talk
between MAPK cascades and JAK-STAT pathways.
MEKK1 is a mammalian Ser/Thr protein kinase initially identified
on the basis of its homology with STE11 and Byr2, the MAPKKKs that
activate the pheromone-responsive MAPK cascade in yeast (25). The
full-length 196-kDa murine MEKK1 is a target for proteolytic cleavage
by caspases. Cleavage of MEKK1 at Asp-874 releases the 91-kDa fragment
containing the COOH-terminal catalytic domain, which renders this
portion constitutively active (26, 27). Expression of the
constitutively active forms of MEKK1 that contain either the catalytic
domain or the COOH-terminal fragment lead to the activation of JNK via
phosphorylation of its upstream kinase, JNKK1/MKK4 (28). MEKK1 also has
the ability to activate ERK, but the effect is less potent (29). These
data suggest MEKK1 to be an upstream kinase in the MAPK cascade. In
addition, MEKK1 is found to participate in the activation of the
transcription factor NF- EGF stimulates activation of Stat3 by phosphorylation on tyrosine and
serine. Since MEKK1 is an upstream kinase of MAPKs that are involved in
serine phosphorylation of Stat3, and MEKK1 itself is also activated by
EGF in various cell type (33, 34), we investigate a possible role of
MEKK1 on Stat3 phosphorylation and activation. To our surprise, the
dominant negative MEKK1 inhibits phosphorylation of Stat3 not only on
serine but also on tyrosine induced either by EGF in COS-1 and HeLa
cells or by PDGF in NIH3T3 cells. On the other hand, a constitutively
activated MEKK1 induces both serine and tyrosine phosphorylation of
Stat3, which is functionally active. We further showed that MEKK1
phosphorylates Stat3 Ser-727 directly in vitro and induces
Stat3 tyrosine phosphorylation via Src and Jaks in vivo. We
suggest that MEKK1 is involved in Stat3 activation by growth factors
via modulating the Jak and Src activities and enhancing serine
phosphorylation either directly or by activation of its downstream
kinases in MAPK pathways.
Construction of Plasmids--
The expression plasmid of Stat3,
pRc/CMV-Stat3 (8), was a gift from Dr. J. E. Darnell (The
Rockefeller University). pRc/CMV-Stat3 mutants were cloned by
substitutions of the phosphorylation site Tyr-705 to Phe (named Y1) or
Ser-727 to Ala (named S1), performed with the QuikChangeTM
site-directed mutagenesis kit (Stratagene). The bacterially expressed glutathione S-transferase (GST)-Stat3 fusion protein and the
point mutant GST-S1, in which Ser-727 of GST-Stat3 was replaced by Ala, were constructed as described previously (12, 22). The full-length Stat3 was also cloned in plasmid pXJ40-GST provided by Dr. E. Manser
from our institute, and the resultant plasmid (mGST-ST3) expresses a
GST-Stat3 fusion protein in mammalian cells. The constitutively active
MEKK1-C (34) was provided by Dr. R. Janknecht (Mayo Foundation). The
point mutant MEKK1-C (KM) was constructed by replacing Lys-1253 to Met
(27) and was confirmed by sequencing. The expression plasmids of
wild-type Src+ (pSGTsrcK+) and the
kinase-inactive Src Antibodies, Growth Factors, and
Inhibitors--
Anti-phospho-Tyr-705 Stat3, anti-phospho-Ser-727
Stat3, and anti-phospho-Tyr-701 Stat1 were purchased from New England
Biolabs. Monoclonal anti-Stat3, anti-EGF-R (activated), and anti-EGF-R antibodies were obtained from Transduction Laboratories. Polyclonal anti-Stat3 (C-20), anti-Stat1 (C-24), anti-MEKK1 (C-22), and anti-Jak2 (C-20 and HR-758) antibodies were purchased from Santa Cruz
Biotechnology. Recombinant mouse MEKK1 enzyme, anti-phosphotyrosine
(4G10), and anti-phospho-Ser-727 Stat1 were purchased from Upstate
Biotechnology, Inc. Anti-phosphoserine monoclonal antibody was obtained
from Sigma, and anti-v-Src (Ab-1) antibody was acquired from Oncogene Science, Inc. EGF was purchased from Genzyme and Upstate Biotechnology, Inc., and PDGF was from Genzyme. EGF-R inhibitor, AG1478, was obtained
from Calbiochem.
Immunoprecipitation/Western Blotting and Immune Complex Protein
Kinase Assay--
Lysis of cells, immunoprecipitation, Western
blotting, and in vitro kinase assays were performed as
described previously (23). The MEKK1-C was immunoprecipitated by an
MEKK1 (C-22) antibody, and MEKK1 kinase assay was performed in a buffer
as described by Xu et al. (37) with minor modifications.
Briefly, the buffer contains 10 mM Hepes (pH 7.3), 10 mM MgCl2, 1 mM benzamidine, 1 mM dithiothreitol, 20 µM cold ATP, and 10 µCi of [ DNA Transfection, CAT Assay, and Electrophoretic Mobility Shift
Assay (EMSA)--
The methods employed were as described previously
(23). CAT assays were normalized with equivalent Dominant Negative MEKK1 Inhibits Stat3 Phosphorylation and
Transcriptional Activity Induced by Growth Factors--
Stat3 is
phosphorylated on Tyr-705 and Ser-727 upon stimulation by growth
factors such as EGF and PDGF. We and others (17, 20, 22, 23) have shown
that JNK and ERK can phosphorylate Stat3 on serine. Since MEKK1 is an
upstream Ser/Thr kinase of JNK and ERK and is also activated by EGF in
various cell types (33, 34), we investigated whether MEKK1 plays a role
in Stat3 phosphorylation stimulated by growth factors. MEKK1-C (KM), a kinase-inactive mutant containing the catalytic domain of murine MEKK1
with a point mutation was cotransfected with Stat3 into the COS-1
cells, which express very little endogenous Stat3. The phosphorylation
of Stat3 in response to EGF stimulation was examined in Western blot
analysis using antibodies that specifically recognize the
phosphorylated forms of Stat3. As shown in Fig.
1A (lane 2, upper
and 2nd panels), EGF induced both tyrosine and serine
phosphorylation of Stat3. Surprisingly, the tyrosine phosphorylation of
Stat3 was significantly inhibited by dominant negative mutant MEKK1-C (KM), but not by its wild-type counterpart, MEKK1-C, which
contains the catalytic domain and is constitutively active (lanes
3 and 4, upper panel). The EGF-induced Ser-727
phosphorylation of Stat3 was also reduced (lane 4, 2nd
panel,), whereas the expression level of Stat3 remains constant
(3rd panel). To confirm this finding, we further tested the
effect of MEKK1-C (KM) on endogenous Stat3 in other cell types. We
found that the tyrosine phosphorylation of the endogenous Stat3
stimulated by EGF was inhibited in HeLa cells (Fig. 1B, upper
panel). Serine phosphorylation of Stat3 in this cell line was also
diminished (Fig. 1B, 2nd panel), although a high
basal level of serine phosphorylation was observed compared with COS-1
cells. Furthermore, PDGF induces tyrosine (10) and serine
phosphorylation of Stat3 in fibroblast cells (23). Similarly, PDGF-induced tyrosine and serine phosphorylations of endogenous Stat3
were also diminished by MEKK1-C (KM) in NIH3T3 cells (Fig. 1C).
One key dogma of Stat3 regulatory role in gene transcription is its
absolute requirement of Tyr-705 phosphorylation that sequentially leads
to dimer formation, nuclear translocation, binding to its recognition
sequence on DNA, and the activation of gene transcription. We next
examined whether the dominant negative MEKK1 was able to inhibit the
transcriptional activity of Stat3 induced by EGF. Stat3 was
cotransfected with a reporter gene containing three hSIE in the absence
or presence of MEKK1-C (KM) and analyzed by CAT assays. Transcriptional
activity of Stat3 was stimulated by EGF to 2-fold (Fig.
2, compare lanes 2 and
6) in the absence of MEKK-1C (KM). In contrast, the
induction was abolished (lanes 7 and 8) in the
presence of MEKK1-C (KM). Together, these results suggest that MEKK1
regulates Stat3 phosphorylation and activation stimulated by growth
factors.
Active MEKK1 Induces Phosphorylation and Activation of
Stat3--
To study further the role of MEKK1 on Stat3, we tested
whether MEKK1 phosphorylates Stat3. The constitutively active murine MEKK1-C and Stat3 were cotransfected in COS-1 cells, and the
phosphorylation of Stat3 was examined. Both Tyr-705 and Ser-727 of
Stat3 were strongly phosphorylated by MEKK1-C in COS-1 cells (Fig.
3A, left panels).
Furthermore, MEKK1-C also induced tyrosine and serine phosphorylation
of the endogenous Stat3 in MCF-7, a human breast cancer cell line (Fig.
3A, right panels). In contrast, phosphorylation of
transfected Stat1 on Tyr-701 in COS-1 cells was not induced by MEKK1-C
or its kinase-inactive mutant, MEKK1-C (KM), although its Ser-727
phosphorylation can be induced by MEKK1-C (Fig. 3B). As a
control, EGF treatment of the cells induced both Tyr-701 and Ser-727
phosphorylation of Stat1.
To demonstrate further the specificity of Stat3 phosphorylation by
MEKK1, varying amounts of MEKK1-C were cotransfected with Stat3 in
COS-1 cells. Stat3 Tyr-705 and Ser-727 phosphorylations can be detected
with as little as 0.5 µg of the transfected MEKK1-C plasmid, and the
phosphorylations were dose-dependent (Fig. 3C). Moreover, similar phosphorylations were also observed by a human MEKK1-C (not shown). These results indicate that active MEKK1 specifically induces both Ser-727 and Tyr-705 phosphorylation of Stat3.
MEKK1-C Stimulates the DNA Binding and Transcriptional Activities
of Stat3--
The functionality of the MEKK1-C-phosphorylated Stat3
was further investigated by measuring its DNA binding and
transcriptional activities. The DNA binding activity was examined using
EMSA with a 32P-labeled oligonucleotide probe hSIE (Fig.
4A). The Stat3-DNA complex
(SIF-A) was observed when COS-1 cells were cotransfected with Stat3 and
MEKK1-C (left panel, lane 4), which could be competed out by
excess unlabeled wild-type hSIE (lane 5) but not by mutant hSIE oligonucleotides M2 and M1 (lanes 6 and 7).
SIF-A was confirmed by the supershift with a specific antibody against
Stat3 (lane 8) but not Stat1 (lane 9).
The transcriptional activity of Stat3 was tested in the absence or
presence of MEKK1-C. The results showed that MEKK1-C increased the
transcriptional activity of Stat3 to 4.1-fold, compared with 6.5-fold
induced by EGF (Fig. 4B, left panel). On the
other hand, the kinase-inactive mutant, MEKK1-C (KM), did not induce
DNA binding and transcriptional activities of Stat3 (data not shown).
To investigate the contribution of the Tyr-705 and Ser-727
phosphorylation to Stat3 activities induced by MEKK1-C, the mutant Stat3 genes bearing either a single mutation on Ser-727 to Ala (S1) or
Tyr-705 to Phe (Y1) were transfected into COS-1 cells. The EMSA and CAT
assay results (Fig. 4, A and B, right
panels) showed that mutation on Tyr-705 completely abrogated the
DNA binding and transcriptional activities of Stat3, whereas mutation
on Ser-727 showed a strong DNA binding but reduced transcriptional
activity. These results demonstrate that in accordance with growth
factor and cytokine stimulation, the MEKK1-C-induced Stat3
phosphorylation results in a functionally active Stat3, capable of
binding to its cognate regulatory sequence and activating
transcription. Phosphorylation of Tyr-705 is absolutely required for
Stat3 activities, whereas Ser-727 phosphorylation plays a dual role, by
positively regulating transcriptional activity but negatively affecting
its DNA binding activity as reported previously (17, 20, 23).
Phosphorylation of Stat3 by MEKK1-C in Vitro--
MEKK1 is a
Ser/Thr kinase. To investigate the possible mechanism of Stat3
phosphorylation induced by MEKK1, we tested whether Stat3 itself is a
direct substrate of MEKK1 by in vitro kinase assay. MEKK1-C
or the kinase-inactive MEKK1-C (KM) was immunoprecipitated with MEKK1
antibody and subjected to in vitro kinase assay in the
presence of [
To examine the Tyr-705 phosphorylation of Stat3 by MEKK1-C, a similar
in vitro kinase assay was performed in the absence of [ Induction of Tyr-705 Phosphorylation of Stat3 by MEKK1-C via Src
and Jak2 but Not EGF-R Tyrosine Kinases--
The above results led us
to investigate the possible tyrosine kinase(s) that may be involved in
the MEKK1-C-induced Stat3 activation. Jaks, Src, and EGF-R tyrosine
kinases known to phosphorylate Stat3 were examined using the
kinase-inactive Jak2 and Src or a specific inhibitor of EGF-R, AG1478.
As shown in Fig. 6A, the tyrosine phosphorylation of Stat3 was induced by the wild-type Jak2 and
Src in the absence of MEKK1-C and further enhanced in the presence of
MEKK1-C. However, the MEKK1-C-induced Tyr-705 phosphorylation was
inhibited by the kinase-inactive Jak2
To test the role of EGF-R on the tyrosine phosphorylation of Stat3 by
MEKK1, transfected COS-1 cells were either left untreated or treated
with EGF, in the presence or absence of AG1478. As shown in Fig.
6B (upper panel), EGF-induced EGF-R activation
was inhibited by AG1478 examined by using an antibody against the activated EGF-R (compare lanes 5 and 6), whereas
MEKK1-C could not induce the activation of EGF-R (lane 2).
Coincidentally, Tyr-705 phosphorylation of Stat3 induced by EGF, but
not MEKK1-C, was inhibited by AG1478 (3rd panel, lanes
6 and 4). These results demonstrated that EGF-R
activation is required for EGF-induced Stat3 phosphorylation. On the
other hand, although EGF activates MEKK1 (33), the active MEKK1 does
not cause a feedback to stimulate EGF-R phosphorylation. More
importantly, MEKK1-C did not activate Stat3 through EGF-R tyrosine kinase.
The function of MEKK1 in mammalian cells has been established as a
MAPKKK that activates JNKK1 (MKK4), and subsequently activates JNK, and
also as an upstream kinase that phosphorylates I EGF stimulates Stat3 phosphorylation on Tyr-705 and Ser-727 (8, 17).
The dominant negative mutant of MEKK1 blocks such phosphorylations,
indicating its essential role in mediating Stat3 activation in EGF
signaling. A compelling question is how MEKK1 is involved in the
EGF-induced phosphorylation of Stat3. It has been reported that
nonreceptor tyrosine kinases Src and Jak1 are required in the Stat3
activation by growth factors, such as colony-stimulating factor-1 and
PDGF (12, 15). We show here that Stat3 tyrosine phosphorylation by
active MEKK1 is abrogated by dominant negative Src and Jak2, and active
MEKK1 does not activate EGF-R, suggesting that MEKK1 functions
downstream of EGF-R but upstream of Src and JAKs (Fig. 6). In our
preliminary results, MEKK1 elevates Jak2 phosphorylation and its kinase
activity (data not shown), implying that Jak2 may be a target of MEKK1.
However, how MEKK1 modulates Jak2 activation remains unclear and
requires further investigation.
Earlier studies have shown that all three subtypes of MAPK are involved
in Stat3 serine phosphorylation. ERK and JNK phosphorylate Stat3 in
response to EGF (17, 20, 22) and various stresses (23), respectively,
whereas p38 regulates Stat3 serine phosphorylation in response to a
combination of IL-12 and IL-2 stimulation (21). We also observed that
MAPKKs, including MEK1, MKK3, MKK6, and MKK7, induce Ser-727
phosphorylation of Stat3 to various degrees (data not shown). Our
results show that MEKK1 phosphorylates Stat3 on Ser-727 directly
in vitro, suggesting that it may phosphorylate Stat3 on
Ser-727 in vivo. However, it may also induce Stat3 serine phosphorylation through the downstream kinases in vivo.
Collectively, these data indicate an extensive cross-talk between STATs
and kinases in the different levels of the MAPK cascades, presumably to
maximize the Stat3 activity.
During the preparation of this manuscript, the small GTPase Rac1 is
reported to regulate Stat3 phosphorylation and activation in response
to EGF stimulation in a very similar manner to MEKK1 (40). Dominant
negative Rac1 inhibits both tyrosine and serine phosphorylation and the
transactivation of Stat3 stimulated by EGF, whereas the active Rac1
induces phosphorylation of both Jak2 and Stat3. Interestingly, MEKK1 is
reported to associate with GTP-bound active Rac1 (33), and its activity
is required for the Rac1-induced JNK activation in EGF signaling (41).
Taken together, it is possible that MEKK1 is involved in the
Rac1-induced Stat3 tyrosine phosphorylation and activation or vice versa.
The possible role of MEKK1 in Stat3 activation in growth factor
signaling is summarized in a simplified model (Fig.
7). Binding of growth factors to their
respective receptors triggers their intrinsic tyrosine kinase activity
that leads to the activation of tyrosine kinases, Src and JAKs, GTPase
Rac1, and its associated MEKK1. The activated MEKK1 further increases
the activity of Src and JAKs and leads to an enhanced tyrosine
phosphorylation of Stat3. On the other hand, MEKK1 activates both the
MKK4-JNK pathway and the ERK pathway, the latter by interacting with
GTP-bound active Ras (42), and Raf-1, MEK1, and ERK2 in a scaffold
complex (43). Therefore, Stat3 serine phosphorylation in EGF signaling may be mediated by multiple kinases in the MAPK pathways including MEKK1, MEK1, ERKs, and JNKs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, which is activated by various
extracellular stimuli, including inflammatory cytokines and stresses
(reviewed in Ref. 30). A key step in the activation of NF-
B is the
phosphorylation of its inhibitor, I
B, by I
B kinases (IKKs) (31).
Transient transfection of the active MEKK1 phosphorylates and
stimulates the kinase activity of IKK
and IKK
, which results in
the phosphorylation of I
B and the release of NF-
B to translocate
into the nucleus (32). This evidence suggests that the function of
MEKK1 is not restricted to the MAPK cascade.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(pSGTsrcK
) (35) were
gifts from Dr. S. A. Courtneidge (Sugen Inc., Redwood City, CA).
Wild-type Jak2 (Jak2+) and the kinase-inactive Jak2
(Jak2
) (36) were obtained from Dr. O. Silvennoinen
(University of Helsinki, Finland). The reporter plasmid pSIE-CAT for
CAT assays was prepared by inserting three copies of the high affinity
sis-inducible element (hSIE) sequence (TTCCCGTAA) upstream
of a c-fos minimal promoter followed by the CAT gene in
plasmid pFOSCAT
56 as described (22).
-32P]ATP, and MEKK1 phosphorylation was
assayed using GST-Stat3 fusion proteins as substrates. For cold
in vitro kinase assay coupled with Western immunoblotting, a
similar procedure as described above was performed except by
incorporating 100 µM cold ATP instead of
[
-32P]ATP in the assay. GST-Stat3 resolved in an
SDS-PAGE and transferred to a PVDF membrane was subjected to Western
immunoblotting with an anti-phosphotyrosine (4G10) and anti-Stat3
(C-20) antibodies. For kinase assay using recombinant MEKK1 and GST or
GST-Stat3 as substrates, cells were transfected with pXJ40-GST or
pXJ40-GST-Stat3, and the expressed GST and GST-Stat3 were immobilized
onto glutathione-Sepharose beads overnight at 4 °C, washed twice
with RIPA, and twice with phosphate-buffered saline. Kinase reaction
was performed in the absence or presence of 0.2 µg of recombinant
MEKK1 at 30 °C for 15 min.
-galactosidase
activity, and the EMSA was performed using hSIE as a probe. For
supershift or competition experiments, Stat3 (C-20) or Stat1 (C-24)
antibodies or molar excess of oligonucleotides were incubated with the
nuclear extracts prior to the addition of radiolabeled hSIE probe.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
MEKK1 is required for the EGF- and
PDGF-induced Stat3 phosphorylation. A, COS-1 cells were
transfected with the expression plasmid of Stat3 in the absence or
presence of 5 µg of MEKK1-C (KM) expression plasmid. Cells were
either left uninduced (U) or stimulated with 100 ng/ml EGF
for 15 min. An aliquot of total cell lysates (30 µg) was resolved on
a 10% SDS-PAGE, transferred to a PVDF membrane, followed by Western
blot analyses with anti-phospho-Tyr-705 Stat3 (pY705 Stat3),
anti-phospho-Ser-727 Stat3 antibody (pS727 Stat3),
anti-Stat3, or anti-MEKK1 (C-22) antibodies. B, HeLa cells
were transfected with vector alone or increasing amounts of MEKK1-C
(KM) expression plasmid (5 µg for lane 3 or 10 µg for
lane 4), and cells were either left uninduced (U)
or stimulated with 100 ng/ml EGF for 5 min. Endogenous Stat3 protein
was immunoprecipitated (IP) from total cell lysates with
anti-Stat3 (C-20) antibody, resolved on a 10% SDS-PAGE, and analyzed
by Western immunoblotting with anti-phosphotyrosine (4G10) antibody
(BLOT: pTyr). The blot was stripped and re-blotted with
anti-phosphoserine (BLOT: pSer), and subsequently with
anti-Stat3 antibody (BLOT: Stat3). An aliquot of total cell
lysates (30 µg) was also resolved and blotted with anti-MEKK1 (C-22)
antibody to analyze to expression of MEKK1-C (KM) protein (TC
BLOT: MEKK1). C, NIH3T3 cells were transfected with
vector alone ( ) or 5 µg MEKK1-C (KM) (+) expression plasmid, and
cells were either left uninduced (U) or stimulated with 50 ng/ml PDGF for 5 min. Immunoprecipitation and analyses of the
endogenous Stat3 protein and analysis of MEKK1-C (KM) expression were
performed as described in B.
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Fig. 2.
MEKK1-C (KM) inhibits EGF-induced Stat3
transcriptional activity. COS-1 cells were transfected with an
empty vector (vec) or expression plasmids encoding Stat3
(St3), either alone or together with 5 or 10 µg of MEKK1-C
(KM), in the presence of the reporter plasmid pSIE-CAT and
pCMV- -gal. Cells were either left uninduced (lanes 1-4)
or stimulated with 100 ng/ml EGF for 6 h (lanes 5-8).
CAT assays were normalized with equivalent
-galactosidase activity
and performed as described under "Experimental Procedures."
Acetylated and non-acetylated forms of
[14C]chloramphenicol were separated by thin layer
chromatography, followed by autoradiography. A representative
autoradiograph of three independent experiments is shown in the
upper panel. The CAT activities were quantified using a
Bio-Rad GS700 imaging densitometer, and the average fold of induction
is illustrated in the graph in the lower panel and indicated
on top of the error bars. Error bars
represent the standard error of the means.
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Fig. 3.
MEKK1-C induces Tyr-705 and Ser-727
phosphorylation of Stat3. A, COS-1 cells were
transfected with the expression plasmid of Stat3 in the absence ( ) or
presence (+) of MEKK1-C expression plasmid (left panel).
MCF-7 cells were transfected with or without MEKK1-C (right
panel). Cells were harvested 40 h after transfection. An
aliquot of total cell lysates (10 µg) was resolved on a 10%
SDS-PAGE, transferred to a PVDF membrane, followed by immunoblotting
with anti-phospho-Tyr-705 Stat3 (pY705 Stat3), or
subsequently with anti-phospho-Ser-727 Stat3 antibody (pS727
Stat3) to analyze the ability of Stat3 phosphorylation by MEKK1-C.
The blots were finally stripped and re-blotted with anti-Stat3 to
analyze the expression of Stat3 protein. B, COS-1 cells were
transfected with the expression plasmid of Stat1 in the absence or
presence of MEKK1-C or MEKK1-C (KM). As a positive control, cells
transfected with Stat1 were stimulated with 100 ng/ml EGF for 15 min
(lane 4). Total cell lysates were resolved and analyzed by
Western immunoblotting using an anti-phospho-Tyr-701 Stat1 (upper
panel), anti-phospho-Ser-727 Stat1 (2nd panel),
anti-Stat1 (3rd panel), and anti-MEKK1 (lower
panel) antibodies as indicated. Anti-phospho-Tyr-701 Stat1
antibody bound nonspecifically to EGF receptor (EGF-R), as
indicated in the figure. C, COS-1 cells were transfected
with Stat3 expression plasmid and varying amounts of MEKK1-C (0.5, 1, 2.5, and 5 µg), followed by Western blotting analysis of total cell
lysates as described for A.
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Fig. 4.
MEKK1-C induces Stat3 DNA binding and
transcriptional activities. A, COS-1 cells were
transfected with expression plasmids encoding Stat3 (St3),
Y1, S1, and/or MEKK1-C. An aliquot of crude nuclear extracts (10 µg)
was subjected to mobility shift DNA binding assay using hSIE as a
probe. SIF-A indicates a complex of hSIE and the homodimer of Stat3.
The competition assay was performed using 100-fold excess of unlabeled
oligonucleotides of either the wild-type (WT) or the mutant
hSIEs (M1 and M2), or with 0.2 µg of anti-Stat3
(C-20) or anti-Stat1 (C-24) antibodies. FP indicates free
probe. B, COS-1 cells were transfected with an empty vector
(vec), Stat3 (St3), or MEKK1-C plus Stat3, Y1, or
S1, in the presence of the reporter plasmid pSIE-CAT and pCMV- -gal.
Cells were left untreated (lanes 1-3 and 5-8)
or treated with 100 ng/ml EGF for 6 h (lane 4) as a
positive control. CAT assays were normalized with equivalent
-galactosidase activity and performed as described under
"Experimental Procedures." A representative autoradiograph of three
independent experiments is shown in the upper panels. The
CAT activities were quantified using a Bio-Rad GS700 imaging
densitometer, and the average fold of induction is illustrated in the
graph in the lower panels and indicated on
top of the error bars. Error bars represent the
standard error of the means.
-32P]ATP using bacterially produced
GST-Stat3 fusion protein (GST-ST3) as a substrate. GST-ST3 was
phosphorylated by MEKK1-C, but not by MEKK1-C (KM) (Fig.
5A), indicating that GST-ST3
is phosphorylated by MEKK1-C, but not likely by any kinase(s) that
may have associated with MEKK1-C. Immunoprecipitated MEKK1-C used
in the kinase assay was shown by Western blot analysis (lower
panel), and equal loading of GST-Stat3 fusion proteins was shown
by staining of the blot with Amido Black (middle panel).
Furthermore, a recombinant mouse MEKK1 enzyme phosphorylated GST-Stat3
fusion protein expressed in COS-1 cells but not the GST protein alone
(Fig. 5B, left panel). The amount of fusion proteins loaded
was determined by staining of the blot with Amido Black (right
panel). To identify the phosphorylation site(s), in
vitro kinase assay was performed using GST-S1 mutant (Ser-727
mutated to Ala) (23) as a substrate. A greatly diminished phosphorylation on GST-S1 compared with GST-ST3 was detected (Fig. 5C).
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Fig. 5.
In vitro phosphorylation of
GST-Stat3 on Ser-727 but not on Tyr-705 by MEKK1-C. A,
COS-1 cells were transfected with an empty vector ( ), MEKK1-C, or the
kinase-inactive MEKK1-C (KM), and cell lysates were immunoprecipitated
(IP) with an anti-MEKK1 (C-22) antibody, followed by
in vitro kinase assay using bacterially expressed GST-Stat3
(GST-ST3) as a substrate in the presence of 10 µCi of
[
-32P]ATP. Proteins resolved on SDS-PAGE were
transferred to nitrocellulose membrane and subjected to autoradiography
(upper panels). Equal loading of Stat3 fusion proteins in
each reaction was confirmed by Amido Black staining of the blot
(middle panels). The lower panels show MEKK1
immunoblots to verify the MEKK1-C immunoprecipitated.
pSTAT3, phospho-Stat3; IP, immunoprecipitation;
BLOT, immunoblot. B, COS-1 cells were transfected
with a mammalian expression vector pXJ40-GST encoding GST
(mGST) or pXJ40-GST-Stat3 encoding GST-Stat3
(mGST-ST3). The expressed GST proteins were immobilized onto
glutathione-Sepharose beads and incubated with 0.2 µg of the
recombinant mouse MEKK1, and an in vitro kinase assay for
MEKK1 was performed (left panel). Right panel
shows the Amido Black-stained blot indicating the amount of mGST or
mGST-ST3 loaded in each lane. C, Similar in vitro
kinase assay as described in A was performed using either
GST-ST3 or a Stat3 point mutant of Ser-727 to Ala (GST-S1)
as substrates, by MEKK1 immunoprecipitated from COS-1 cells transfected
with either empty vector (
) or MEKK1-C. D, cells were
transfected with an empty vector (
), MEKK1-C, MEKK1-C (KM), or
wild-type Src+ as a positive control. MEKK1-C and Src were
immunoprecipitated with their respective antibodies, and a cold
in vitro kinase assay was performed as described above
except in the presence of 100 µM cold ATP instead of
[
-32P]ATP, using bacterially expressed GST-ST3 as
substrate. Western immunoblotting was performed with an
anti-phosphotyrosine antibody, 4G10 (pTyr Stat3, upper
panel). The blot was stripped and blotted with an anti-Stat3
(C-20) antibody (lower panel).
-32P]ATP in the reaction. Instead, it contained 100 µM cold ATP, and the reaction mixtures were subjected to
Western blot analysis using the anti-phosphotyrosine 705 Stat3
antibody. The results revealed that whereas the positive control, Src,
immunoprecipitated from the Src-transfected cells, was able to
phosphorylate GST-ST3 on tyrosine, MEKK1-C failed to do so (Fig.
5D). These findings support the hypothesis that MEKK1, a
Ser/Thr kinase, is capable of phosphorylating Stat3 on Ser-727 directly
in vitro but on Tyr-705 via an indirect mechanism.
or
Src
. Expressions of Stat3, MEKK1-C, Jak2, and Src are
shown in lower panels. These results suggest that Jak2 and
Src are required for the Stat3 Tyr-705 phosphorylation by MEKK1-C.
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Fig. 6.
Tyr-705 phosphorylation of Stat3 induced by
MEKK1-C via Src and Jak2, but not EGF receptor. A,
COS-1 cells were transfected with expression vectors encoding Stat3,
MEKK1-C, Jak2+, Jak2 , Src+, or
Src
as indicated. An aliquot of total cell lysates was
subjected to Western immunoblotting with antibodies as indicated,
including anti-Src (Ab-1) and anti-Jak2 (C-20) antibodies.
B, COS-1 cells were transfected with Stat3 alone or together
with MEKK1-C and were either left uninduced (lanes 1 and
2) or treated for 30 min with 20 µM AG1478,
the inhibitor of EGF receptor (lanes 3 and 4). As
controls, cells were induced with 100 ng/ml EGF for 15 min (lane
5) or pre-treated with 20 µM AG1478 for 15 min
followed by EGF for another 15 min (lane 6). An aliquot of
total cell lysates was resolved on a 10% SDS-PAGE followed by Western
immunoblotting with antibodies indicated on the right.
M, MEKK1-C; E, EGF.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B kinases leading to
the activation of NF-
B by using expression of either the catalytic
domain of MEKK1 or a 672-amino acid COOH-terminal fragment (38, 39). In
this study, we used a similar strategy and identified the JAK-STAT
pathway as another target of MEKK1. We demonstrate that MEKK1 regulates
Stat3 activity via inducing a unique dual phosphorylation on both
Tyr-705 and Ser-727, which leads to a functionally active Stat3. Since
the Tyr-705 phosphorylation is a prerequisite for Stat3 activity, the
ability of MEKK1, a Ser/Thr kinase, to stimulate tyrosine
phosphorylation of Stat3 is rather remarkable. However, we consider
this effect specific and unlikely to be due to the overexpression of
the active MEKK1. We observed that a low amount of MEKK1-C (0.5 µg)
already significantly induces Tyr-705 phosphorylation of Stat3 (Fig.
3C). In addition, full-length MEKK1 also induces tyrosine
phosphorylation of Stat3, albeit weaker (data not shown). However, none
of the other kinases in the MAPK cascades we have examined, including
MAPKKs MKK3, MKK6, MKK7, and MEK1, can induce Tyr-705 phosphorylation
of Stat3 when overexpressed (data not shown). We also detected Stat5
phosphorylation by MEKK1-C (not shown), but not Stat1 (Fig.
3B), under the same conditions. The specificity was also
supported by the observation of phosphorylation of endogenous Stat3 in
MCF-7 (Fig. 3A). Our data suggest that MEKK1 selectively
induces phosphorylation and activation of certain members of STAT family.
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Fig. 7.
Proposed model for the role of MEKK1 in Stat3
activation in EGF signaling. MEKK1 is activated by EGF via Rac1.
Active MEKK1 further stimulates Src and Jak kinases and leads to
Tyr-705 phosphorylation of Stat3. MEKK1 also activates ERK and JNK MAPK
cascades, and Stat3 Ser-727 can be phosphorylated by multiple kinases
in the MAPK cascades including MEKK1, MEK1, and ERK/JNK.
Although JNK and NF-B reside in two different signaling pathways and
have distinct downstream targets, both can be activated by inflammatory
cytokines, such as tumor necrosis factor-
and interleukin-1, as well
as environmental stresses (44), and therefore can be coordinately
activated via MEKK1. However, we previously observed that only Ser-727,
but not Tyr-705 phosphorylation, was induced by tumor necrosis
factor-
and various stress treatments, suggesting that activation of
MEKK1 by these stimuli does not lead to activation of Stat3. On the
other hand, MEKK1 activated by EGF results in the activation of Stat3,
JNK, and ERK (28, 29). Therefore, MEKK1 responds to different
extracellular stimuli by specifically activating a subset of the
downstream signaling molecules to exert its function in controlling a
wide range of cellular responses. Thus, MEKK1 may represent an
important mediator in the cellular signaling network.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. R. Janknecht for the
constitutively activated MEKK1-C plasmid; J. E. Darnell for
pRC/CMV-Stat3; S. A. Courtneidge for wild-type
pSGTsrcK+ and the kinase-inactive pSGTsrcK;
O. Silvennoinen for Jak2; and Y. Zhang and S.-C. Lin for human MEKK1-C
expression plasmids. We thank Dr. B. C. Low, Dr. V. Novotny, and
T. Zhang for the critical reading of the manuscript and R. Tham
and R. Chng for photography.
![]() |
FOOTNOTES |
---|
* This work was supported by the National Science and Technology Board of Singapore.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.
To whom correspondence should be addressed: Institute of
Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore. Tel.: 65-874-3795; Fax: 65-779-1117; E-mail:
mcbcaoxm@imcb.nus.edu.sg.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M007592200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
STAT, signal
transducers and activators of transcription;
JAK, Janus kinase;
MEKK1, MEK kinase 1;
MAPKK, mitogen-activated protein kinase kinase;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated
kinase;
JNK1, c-Jun NH2-terminal kinase 1;
EGF, epidermal
growth factor;
EGF-R, EGF receptor;
PDGF, platelet-derived growth
factor;
IL-6, interleukin-6;
GST, glutathione S-transferase;
EMSA, electrophoretic mobility shift assay;
CAT, chloramphenicol
acetyltransferase;
PAGE, polyacrylamide gel electrophoresis;
IKK, IB
kinases;
PVDF, polyvinylidene difluoride.
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