Cross-talk between JNK/SAPK and ERK/MAPK Pathways
SUSTAINED ACTIVATION OF JNK BLOCKS ERK ACTIVATION BY MITOGENIC FACTORS*
Ying H. Shen,
Jakub Godlewski,
Jun Zhu,
Pradeep Sathyanarayana,
Virna Leaner
,
Michael J. Birrer
,
Ajay Rana and
Guri Tzivion
From the
Cardiovascular Research Institute, Division of Molecular Cardiology, the
Texas A&M University System Health Science Center, College of Medicine,
Temple, Texas 76504 and
Cell and Cancer
Biology Department, NCI, National Institutes of Health, Rockville, Maryland
20850
Received for publication, March 29, 2003
, and in revised form, April 28, 2003.
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ABSTRACT
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Mixed lineage kinases (MLKs) are a family of serine/threonine kinases that
function in the SAPK signaling cascade. MLKs activate JNK/SAPK in
vivo by directly phosphorylating and activating the JNK kinase SEK-1
(MKK4 and -7). Importantly, the MLK member MLK3/SPRK has been shown recently
to be a direct target of ceramide and tumor necrosis factor-
(TNF-
) and to mediate the TNF-
and ceramide-induced JNK
activation in Jurkat cells. Here we report that MLK3 can phosphorylate and
activate MEK-1 directly in vitro and also can induce MEK
phosphorylation on its activation sites in vivo in COS-7 cells.
Surprisingly, this induction of MEK phosphorylation does not result in ERK
activation in vivo. Rather, in cells expressing active MLK3, ERK
becomes resistant to activation by growth factors and mitogens. This
restriction in ERK activation requires MLK3 kinase activity, is independent of
Raf activation, and is reversed by JNK pathway inhibition either at the level
of SEK-1, JNK, or Jun. These results demonstrate that sustained JNK activation
uncouples ERK activation from MEK in a manner requiring Jun-mediated gene
transcription. This in turn points to the existence of a negative cross-talk
relationship between the stress-activated JNK pathway and the
mitogen-activated ERK pathway. Thus, our findings imply that some of the
biological functions of JNK activators, such as TNF-
and ceramide, may
be attributed to their ability to block cell responses to growth and survival
factors acting through the ERK/MAPK pathway.
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INTRODUCTION
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Mixed lineage kinases
(MLKs)1 were
identified a decade ago as dual specificity kinases, i.e. kinases
with both serine/threonine and tyrosine kinase activity
(14).
Although the tyrosine kinase activity of MLKs has not yet been reported, MLKs
contain the tyrosine kinase signature motif, in addition to the
serine/threonine kinase motif (for review see Ref.
5). Soon after their
identification, several groups
(69)
independently demonstrated that MLK family members can directly phosphorylate
and activate the JNK upstream kinase SEK-1 in vitro and induce JNK
activation in vivo. These findings classified MLK family members as
mitogen-activated protein kinase kinase kinases (MAPKKKs) in the
stress-activated signaling cascade (for review see Refs.
5 and
10). The validity of this
classification is strongly substantiated by the recent identification of
scaffold proteins that bind several members of the stress-activated protein
kinase cascade (i.e. MLK, SEK-1, and JNK) and augment the signaling
through the cascade
(1113).
The identification of an upstream activator(s) of MLK family members turned
out, however, to be a much more complicated task. Besides the signature kinase
motif, MLK members contain a Cdc42/Rac-interactive binding domain, the
presence of which pointed to the possibility that Cdc42 and Rac may be
upstream regulators of MLKs
(14,
15). Additional work is
required to determine more conclusively the role of Cdc42 or Rac in MLK
activation.
A breakthrough in understanding MLK activation comes from two recent
studies (16,
17) that used
Drosophila MLK as a model. Together, these studies provide genetic
and biochemical evidence positioning MLK as a critical JNK activator and
define TNF-
and ceramide as potent natural activators of MLKs. These
findings are in agreement with other studies
(1820)
demonstrating a critical role of MLKs in JNK-mediated neuronal apoptosis.
Besides SEK-1 (MKK4 and -7), no other MLK targets have been defined clearly
(5). Few reports indicate that
MLK members can activate the ERK/MAPK pathway, possibly through MEK-1
activation, and suggest an oncogenic potential of MLK
(21). However, the details of
MEK activation and the oncogenic potential have not been determined.
The various MAPK cascades (e.g. ERK1/2, JNK, p38, and ERK5) are
often portrayed in the literature as linear cascades, and indications for
cross-talk between the various cascades are limited
(5,
10). In this respect, the
present study examines the consequences of MLK3 overexpression on the ERK/MAPK
pathway and its subsequent response to mitogenic stimuli. We find that MLK3
can phosphorylate and activate MEK-1 both in vitro and in
vivo. MEK activation in vivo, however, is uncoupled from ERK
activation. Moreover, in cells expressing active MLK3, ERK becomes resistant
to activation by mitogens. This restriction in ERK activation involves the
SEK-1-JNK-Jun cascade, as demonstrated by the ability of specific inhibitors
of the pathway to reverse the blockage of ERK activation. Our results
demonstrate negative cross-talk between the stress-activated MLK-SEK-JNK-Jun
pathway and the ERK/MAPK pathway, and suggest that sustained activation of the
JNK pathway can result in the attenuation of the mitogen-activated ERK
pathway.
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EXPERIMENTAL PROCEDURES
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cDNA Constructs, Antibodies, and Kinase
InhibitorsM2-FLAG-MLK3 and M2-FLAG-kinase-dead MLK3 (K144A) were
in the pRK-5 mammalian expression vector (for construction details see Ref.
3)). myc-Raf-1 and HA-ERK-1
were in the pMT2 mammalian expression vector
(6,
22). Wild-type c-Jun and the
dominant negative c-Jun variants TAM-67 (deletion mutant lacking the
transactivating domain) and DBM-3 (variant containing mutations in the DNA
binding domain) were in pCMV vector
(2325).
Wild-type EE epitope-tagged MEK-1 and constitutively active MEK-1
(S218D/S222D) were in the pCDNAI vector
(26). Constitutively active
c-Raf (GST-Bxb-Raf) and dominant negative HA-SEK-1-AL (S220A and T224L) were
in the pEBG vector (6,
27,
28).
Phosphospecific antibodies for the active forms of ERK, MEK, and c-Jun and
corresponding antibodies for the non-phosphorylated forms were from Cell
Signaling Technology (Beverly, MA). Antibodies against HA, Myc, and the EE
epitope were produced from 12CA5, 9E10, and EE hybridoma cell lines,
respectively. Anti-M2-FLAG epitope antibody was purchased from Sigma. JNK
inhibitors, JNK inhibitor I (a cell-permeable peptide inhibitor), and JNK
inhibitor II (an ATP competitive cell-permeable inhibitor) were from
Calbiochem. The MLK inhibitor CEP-11004 was a kind gift from Cephalon.
Cell Culture and TransfectionCOS-7 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For
transient expression of proteins, cells were transfected using LipofectAMINE
(Invitrogen) according to the manufacturer's instructions (detailed in the
figure legends). Details for cell stimulation and treatment with inhibitors
are provided in the figure legends.
Cell Extraction and Protein PurificationCells were lysed
for 30 min in ice-cold extraction buffer containing 50 mM Tris-Cl
(pH 7.5), 100 mM NaCl, 1% Triton X-100, 1 mM
dithiothreitol, 1 mM EDTA, 1 mM EGTA, 2 mM
Na3VO4, 50 mM
-glycerophosphate, and a
protease inhibitor mixture (Amersham Biosciences). For immunoprecipitation,
cleared cell lysates were incubated at 4 °C for 90 min with the
appropriate antibody precoupled to protein A/G-agarose beads (Santa Cruz
Biotechnology). The beads were washed twice with extraction buffer, twice with
extraction buffer containing 0.5 M LiCl, and twice with kinase
assay buffer (40 mM Tris-Cl (pH 7.5), 0.1 mM EDTA, 5
mM MgCl2, and 2 mM dithiothreitol). Proteins
were eluted directly in SDS sample buffer for Western blot analysis or were
assayed for kinase activity, as indicated in the figure legend.
Kinase AssaysRaf kinase activity was assayed as described
previously (22). Briefly,
following Myc immunoprecipitation, myc-Raf containing beads were incubated for
20 min at 30 °C in kinase assay buffer (100 µl final volume)
supplemented with 100 µM ATP, 10 µCi of
[
-32P]ATP, and 0.3 µg of prokaryotic recombinant
GST-MEK-1. Two micrograms of prokaryotic recombinant kinase inactive ERK
(K52R) were added, and the samples were incubated for an additional 30 min.
Samples were separated on 10% SDS-PAGE and transferred to PVDF membranes.
32P incorporation into ERK was quantified by PhosphorImager (PI)
analysis (Storm, Amersham Biosciences). Data are presented PI x
103. myc-Raf recovery was determined by Myc immunoblotting. For
assessment of ERK kinase activity, following HA immunoprecipitation, HA-ERK
containing beads were incubated for 10 min at 30 °C in kinase assay buffer
(50 µl final volume) supplemented with 100 µM ATP, 10 µCi
of [
-32P]ATP, and 10 µg of myelin basic protein (MBP,
Sigma). Samples were separated on 12% SDS-PAGE and transferred to PVDF
membranes. 32P incorporation into MBP was quantified by
PhosphorImager analysis (data are presented PI unit x 103).
ERK recovery was determined by immunoblotting with anti-HA or anti-ERK
antibodies, as detailed in the figure legends. To assess MLK3 kinase activity
and to determine the effect of MLK3 on the Raf-MEK-ERK-MBP cascade in
vitro, the combination of proteins indicated in the figure legends was
incubated for the indicated times at 30 °C in kinase assay buffer (100
µl final volume) supplemented with 100 µM ATP and 10 µCi
of [
-32P]ATP. Samples were separated on 8.5% SDS-PAGE;
samples containing MBP were separated on 12% SDS-PAGE. Separated proteins were
transferred to PVDF membranes. Kinase activity was analyzed by autoradiography
and PhosphorImaging. Phosphospecific antibodies recognizing the activated
forms of ERK, MEK, and c-Jun were used for determining in vivo
activation levels of ERK, MEK, and Jun, respectively.
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RESULTS
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Expression of Active MLK3 in COS-7 Cells Attenuates ERK Activation by
Growth Factors and Mitogens without Affecting Raf ActivationMLK3
functions as a MAPKKK in the stress-activated JNK pathway; however, a few
reports suggest that MLK3 may also affect the ERK/MAPK pathway
(21,
29,
30). To determine the effect
of MLK3 on the ERK/MAPK pathway, ERK-1 was co-expressed together with
wild-type MLK3 or a kinase-dead MLK3 mutant, MLK3 (K144A), and the basal and
mitogen-stimulated ERK-1 kinase activity was examined
(Fig. 1, A and
B). In serum-deprived COS-7 cells, expression of active
MLK3 results in a slight, reproducible activation of basal ERK kinase activity
and ERK phosphorylation (Fig.
1B, compare lane 1 with 6,
Fig. 2E, compare
lane 1 with 3, and Fig.
3, compare lane 1 with 3). This activation
depends on MLK3 kinase activity, because it is not observed in cells
expressing the kinase-dead MLK3 mutant. In contrast to basal ERK activation,
MLK3 expression almost completely attenuates ERK activation in response to EGF
and PMA (Fig. 1A,
compare lanes 2 and 3 with lanes 6 and 7
and Fig. 1B, compare
lane 2 with 7). This attenuation depends on MLK3 kinase
activity, because ERK activation is not affected in cells expressing the
kinase-dead MLK3 mutant (Fig. 1, A
and B). Treatment of cells with the phosphatase inhibitor
calyculin A fails to reverse the MLK3 effect on ERK activation
(Fig. 1, A and
B), indicating that activation of phosphatases sensitive
to calyculin A were not responsible for the observed inhibition of ERK
activation.

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FIG. 1. Expression of active MLK3 attenuates ERK activation in vivo
downstream of Raf. A, COS-7 cells in 10-cm plates were
co-transfected with 1.5 µg of pMT2 vector expressing HA-ERK-1 and either
3.5 µg of empty pRK-5 vector (lanes 14), pRK-5 vector
expressing wild-type MLK3 (wt, lanes 58), or pRK-5 vector
expressing kinase-dead MLK3 (kd, lanes 912). Twenty four hours
after transfection, cells were deprived of serum for 18 h and were treated
with either vehicle (, lanes 1, 5, and 9), 100 ng/ml
EGF (EGF, lanes 2, 6, and 10), 200 nM PMA
(PMA, lanes 3, 7, and 11), or 150 nM calyculin A
(cal A, lanes 4, 8, and 12) for 10 min. Cells were lysed,
and ERK kinase activity in the HA immunoprecipitates was assayed as detailed
under "Experimental Procedures." Presented are an autoradiogram
and a bar graph showing 32P incorporation into MBP, a Western blot
showing HA-ERK recovery, and an M2-FLAG immunoblot showing M2-MLK3 expression
in total cell extracts. B, COS-7 cells transfected as in A,
were deprived of serum for 18 h and treated with either vehicle (lanes 1,
6, and 11), 100 ng/ml EGF (lanes 2, 7, and
12), or 150 nM calyculin A (lanes 3, 8, and
13) for 10 min, or with 150 nM calyculin A for 10 min
followed by 100 nM EGF for 10 min (lanes 4, 9, and
14) or with 150 nM calyculin A for 20 min (lanes 5,
10, and 15). HA-ERK kinase activity was determined as in
A, and the level of ERK phosphorylation at its activation sites was
determined by immunoblotting with phosphospecific antibodies for ERK
activation sites (Cell Signaling Technology). Presented are an autoradiogram
showing 32P incorporation into MBP (top panel), a Western
blot showing HA-ERK phosphorylation at its activation sites (middle
panel), and a Western blot showing HA-ERK recovery (bottom
panel). C, COS-7 cell were co-transfected with 1.5 µg of pMT2
vector expressing myc-Raf-1 and either 3.5 µg of empty pRK-5 vector,
pRK-5-MLK3, or pRK-5-MLK3 (kd), as indicated. Cells were stimulated as in
A, and Raf kinase activity in Myc-immunoprecipitates was assayed as
detailed under"Experimental Procedures."Presented are an
autoradiogram and a bar graph showing 32P incorporation in ERK, a
Western blot showing myc-Raf recovery, and an M2-FLAG immunoblot showing
M2-MLK3 expression in total cell extracts.
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FIG. 2. MLK3 does not inhibit the Raf-MEK-ERK-MBP phosphorylation cascade in
vitro, rather MLK3 activates MEK in vitro and induces MEK
phosphorylation at its activation sites in vivo. A, 50
ng of active Raf-1, produced in sf9 cells by co-infection with active Ras and
Src, was incubated in kinase assay buffer at 30 °C with either vehicle, 1
µg of wild-type, or kinase-dead MLK3, as indicated. Following a 15-min
incubation, vehicle, 500 ng of recombinant GST-MEK-1, and 2 µg of
kinase-dead recombinant GST-ERK-1 (kd) were added at the indicated
combinations, and the incubation was continued for additional 15 min. The
protein samples were separated on 8.5% SDS-PAGE and analyzed by
autoradiography. Note that the samples in lanes 2 and
610 do not contain Raf. B, the experiment was
conducted as in A, except that recombinant wild-type GST-ERK-1 was
used instead of kinase-dead ERK-1. MBP was added after the second 15-min
incubation, and the samples were incubated for an additional 10 min. Proteins
were separated using 12% SDS-PAGE and analyzed by autoradiography. C,
2 µg of recombinant GST-MEK-1 was incubated in kinase buffer with vehicle
or 1 µg of MLK3 for 15 min, followed by addition of recombinant kinase-dead
GST-ERK-1. After an additional 15 min of incubation, protein samples were
resolved on 8.5% SDS-PAGE, transferred to PVDF membranes, and analyzed for
kinase activity by autoradiography (top and middle panel)
and immunoblotting with phospho-MEK antibody (bottom panel).
D, COS-7 cells were transfected with 1.5 µg of pCDNAI vector
expressing EE-tagged MEK-1 and either 3.5 µg of empty pRK-5 vector,
pRK-5-MLK3, or pRK-5-MLK3 (kd). Cells were treated with 100 ng/ml EGF and
lysed, and the EE-MEK was immunoprecipitated. Phosphorylation of EE-MEK was
determined by immunoblotting with MEK activation site phosphospecific
antibodies (top panel), and MEK recovery was determined by MEK
immunoblotting (bottom panel). E, COS-7 cells were
co-transfected with 1.5 µg of pMT2-HA-ERK-1 and either 3.5 µg of empty
pRK-5 vector or pRK-5-MLK3, pRK-5-MLK3 (kd), or pCDNAI vector expressing
active MEK-1 (MEK-1-DD). ERK-1 phosphorylation in HA immunoprecipitates was
determined by immunoblotting with phosphospecific ERK antibodies (top
panel), and HA-ERK recovery was determined by immunoblotting with ERK-1
antibody (bottom panel).
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FIG. 3. Dominant negative SEK-1 reverses the effect of MLK3 on ERK
activation. COS-7 cells were transfected with 1.5 µg of pMT2-HA-ERK-1
vector together with 1.5 µg of pRK-5 empty vector and 2 µg of pEBG empty
vector (lanes 1 and 2), 1.5 µg of pRK-5-MLK3 and 2 µg
of pEBG empty vector (lanes 3 and 4), 1.5 µg of
pRK-5-MLK3 and 2 µg of pEBG-SEK-1-AL (dn, lanes 5 and 6),
or 3.5 µg of pEBG-GST-Bxb-Raf (lanes 7 and 8). Twenty
four hours after transfection, cells were deprived of serum for 18 h and
treated with vehicle or 100 ng/ml EGF as indicated. A, HA-ERK kinase
activity was determined using MBP as the substrate, as in
Fig. 1A. Presented are
an autoradiogram showing MBP phosphorylation (top panel) and an ERK
immunoblot showing HA-ERK recovery (bottom panel). B, HA-ERK
activation in same samples was determined by assaying HA-ERK
autophosphorylation in vitro (top panel) and HA-ERK
phosphorylation at its activation sites in vivo (bottom
panel). C, M2-FLAG-MLK3 expression in total cell lysates was
determined by M2 immunoblotting. IP, immunoprecipitation.
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To examine whether the inhibition of ERK activation was due to an
inhibition of Raf activation, Myc-tagged c-Raf-1 was co-expressed with the two
MLK3 variants, and the kinase activity of Raf was measured in a coupled kinase
assay (Fig. 1C).
Expression of active or inactive MLK3 does not affect basal or
mitogen-stimulated Raf kinase activity, suggesting that expression of active
MLK3 uncouples the activation of ERK from Raf.
MLK3 Does Not Inhibit the Raf-MEK-ERK-MBP Phosphorylation Cascade in
Vitro, Rather MLK3 Functions as a Potent MEK Kinase, Both in Vitro and in
VivoTo examine whether MLK3 directly inhibits the kinase
activities of Raf, MEK, or ERK, we tested the effect of MLK3 on the
Raf-MEK-ERK phosphorylation cascade in vitro
(Fig. 2, A and
B). We find that under in vitro conditions, MLK3
does not affect the ability of Raf to phosphorylate MEK-1 or to activate
MEK-1, and it does not affect the ability of MEK-1 to phosphorylate ERK-1
(Fig. 2A). In
addition, MLK3 does not affect the ability of MEK-1 to activate ERK-1, and it
does not affect ERK-1 kinase activity (Fig.
2B).
In contrast to the inhibitory effect of MLK3 on the ERK pathway in
vivo, our experiments show that in vitro, MLK3 phosphorylates
MEK-1 and activates its kinase activity as potently as Raf
(Fig. 2, A and
B). These findings demonstrate that MLK3 can function as
a MAPKKK also in the ERK/MAPK cascade. Because MEK-1-phosphospecific
antibodies that recognize the doubly phosphorylated MEK-1 react with MEK-1
phosphorylated by MLK3 (Fig.
2C), we infer that MEK-1 sites phosphorylated by MLK3 are
similar to the sites phosphorylated by Raf.
MLK3 also induces MEK-1 phosphorylation in vivo, in a manner
depending on its kinase activity (Fig.
2D); however, this does not translate to ERK activation
(Fig. 2E). These
results indicate that, in cells expressing active MLK3, there is an uncoupling
of ERK and MEK kinase activities.
Dominant Negative SEK-1 Reverses the Effect of MLK3 on ERK
ActivationBecause MLK3 apparently phosphorylates both MEK-1 and
SEK-1, we wanted to determine which of these two MLK3-associated activities is
responsible for inhibition of ERK-1 activation. To exclude the possibility
that strong activation of MEK-1 and/or ERK-1 results in a feedback that blocks
ERK-1 activation by mitogens, we examined the effects of the active forms of
MEK-1 (MEK-1-DD, Fig.
2E, lanes 7 and 8) and Raf-1 (Bxb-Raf,
Fig. 3, lanes 7 and
8) on ERK-1 activity. These experiments demonstrate that a
constitutive activation of the Raf-MEK-ERK pathway does not result, by itself,
in ERK inhibition. To examine the possibility that MLK3 attenuates ERK
activation through activation of SEK-1, we co-expressed dominant negative
SEK-1 (SEK-1-AL) and MLK3, and we assayed ERK activation induced by EGF
(Fig. 3). Co-expression with
SEK-1-AL completely reverses the effect of MLK3 on ERK activation
(Fig. 3, A and
B, compare lanes 3 and 4 with lanes
5 and 6). This reversal occurs without affecting MLK3 expression
levels (Fig. 3C).
Although this reversal is most likely due to inhibition of endogenous SEK-1
activity, it is important to note that SEK-1-AL may also inhibit MLK3 by
out-competing other substrates. This possibility is addressed below by using
inhibitors for SEK-1 effectors.
JNK Inhibitors and the MLK3 Inhibitor CEP-11004 Reverse the Effect of
MLK3 on ERK ActivationTo determine which of the components of the
JNK pathway are required for the MLK3-induced ERK inhibition, and to exclude
the possibility that the SEK-1 dominant negative acts by merely inhibiting the
MLK3 kinase activity, we tested the effect of two reportedly specific JNK
inhibitors on the ability of MLK3 to inhibit ERK activation
(Fig. 4, A and
B). Incubation of MLK3-expressing COS-7 cells with two
distinct JNK inhibitors (JNK inhibitor I, a cell-permeable peptide inhibitor,
and JNK inhibitor II, an ATP competitive cell-permeable inhibitor) for 24 h
prior to mitogenic stimulation completely reverses MLK3-induced ERK inhibition
(Fig. 4A, compare
lanes 5 and 6 with lanes 711;
Fig. 4B, compare
lanes 3 and 4 with lanes 9 and 10). This
reversal happens without affecting basal ERK activity or the magnitude of ERK
activation by EGF in cells that do not express MLK3
(Fig. 4A, compare
lanes 1 and 2 with lanes 3 and 4).

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FIG. 4. MLK3 and JNK kinase activities are required for MLK3-induced ERK
inhibition. A, COS-7 cells were co-transfected with 1.5 µg of
pMT2-HA-ERK-1 vector and either 3.5 µg of pRK-5 empty vector (lanes
14) or pRK-5-MLK3 (lanes 511). Twenty four hours
after transfection, cells were deprived of serum for 18 h in the presence or
absence of the indicated concentrations of JNK inhibitors I and II, as
indicated. Cells were treated with 100 ng/ml EGF for 10 min, as indicated, and
HA-ERK was immunoprecipitated. ERK activation in the immunoprecipitates was
determined by phospho-ERK immunoblotting (top panel); HA-ERK recovery
was determined by ERK immunoblotting (middle panel), and M2-FLAG-MLK3
expression in total cell lysates was determined by M2-FLAG immunoblotting
(bottom panel, note that MLK-3 expressed in COS-7 cells appears as a
double band in which the top band is the phosphorylated, active form of MLK3).
B and C, COS-7 cells were co-transfected with 1.5 µg of
pMT2-HA-ERK-1 (B) or 1.5 µg of pCDNI-EE-MEK-1 (C) and
either 3.5 µg of pRK-5-wild-type or kinase-dead MLK3, as indicated. Cells
were treated as in A with either 25 µM JNK inhibitor I
(JNKi-I, lane 9), 100 µM JNK inhibitor II (JNKi-II,
lane 10), or 500 nM CEP-11004 (cep, lanes 7 and
8), and then stimulated with 100 ng/ml EGF for 10 min.
Phosphorylation of HA-ERK in HA-immunoprecipitates (B) and
phosphorylation of MEK in EE-immunoprecipitates (C) was determined by
immunoblotting with phospho-ERK and phospho-MEK, respectively (top
panel). Recovery of HA-ERK and EE-MEK was determined by immunoblotting
with ERK and MEK, respectively (middle panel). M2-FLAG-MLK3
expression in total cell lysates was determined by M2-FLAG immunoblotting
(bottom panel).
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To support the notion that MLK3 kinase activity is required for
MLK3-induced ERK inhibition, the MLK-specific inhibitor CEP-11004
(17,
31) was used to block MLK3
kinase activity (Fig.
4B). CEP-11004 completely reverses MLK3-induced ERK
inhibition, without affecting basal ERK activity
(Fig. 4B, compare
lanes 3 and 4 with lanes 7 and 8).
In parallel experiments, the effects of the JNK inhibitors and CEP-11004
were examined on the ability of MLK3 to induce MEK phosphorylation
(Fig. 4C). Because JNK
peptide inhibitor I reverses ERK inhibition without affecting the ability of
MLK3 to induce MEK-1 phosphorylation (Fig.
4, B and C, lane 9), we conclude that
the inhibition in ERK activation is independent of MEK activation but requires
JNK activity. In addition, these results demonstrate that MLK3-induced MEK
phosphorylation is independent of the ability of MLK3 to activate JNK.
MLK3-induced MEK phosphorylation, however, requires MLK3 kinase activity,
because CEP-11004 blocks MLK3-induced MEK phosphorylation
(Fig. 4C, lane
8). It is important to note that JNK inhibitor II appears not to
specifically target JNK, because it inhibits both MLK3 autophosphorylation
(Fig. 4A, compare
lanes 5 and 6 with lanes 79) and
MLK3-induced MEK phosphorylation (Fig.
4C, compare lanes 3 and 4 with
lanes 7 and 8).
c-Jun-mediated Gene Transcription Is Required for the Ability of MLK3
to Block ERK ActivationTo examine whether JNK directly affects ERK
activation or whether the MLK3-induced ERK inhibition requires c-Jun-mediated
gene transcription, we co-expressed dominant negative forms of c-Jun with MLK3
and tested their effect on the MLK3-induced ERK inhibition
(Fig. 5). Two forms of dominant
negative c-Jun
(2325),
one lacking the amino-terminal transactivation domain (TAM-67) and the second
impaired in its DNA binding (DBM-3), completely reversed MLK3-induced ERK
inhibition (Fig. 5A,
compare lanes 36 with lanes 912). This
reversal happens without affecting MLK3 expression
(Fig. 5B, bottom
panel) or MLK3-induced Jun phosphorylation
(Fig. 5B, top
panel). In contrast, expression of wild-type c-Jun fails to reverse ERK
inhibition and perhaps, if at all, enhances the ability of MLK3 to block ERK
activation (Fig. 5A,
compare lanes 3 and 4 with 7 and 8). These
results demonstrate that c-Jun-mediated gene transcription is critical for the
ability of MLK3 to attenuate ERK activation in response to EGF. Notably,
expression of active MLK3 results in a significant increase of c-Jun
phosphorylation on its activation sites
(Fig. 5B, compare
lanes 7, 8, 11, and 12 with lanes 15, 16, 19, and
20). This increased phosphorylation is observed on both wild-type and
DBM-3 c-Jun but not on TAM-67 c-Jun lacking the transactivation amino-terminal
part (Fig. 5B).
Together, the results presented in Fig.
5 demonstrate that, even under conditions in which MLK3 is active
and is able to activate the SEK-1-JNK-c-Jun pathway, inhibition of c-Jun
reverses MLK3-induced MEK-ERK uncoupling. This indicates that MLK3 restricts
ERK activation at a point downstream of c-Jun.

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FIG. 5. Jun transcriptional activity is required for MLK3-induced ERK
inhibition. COS-7 cells were transfected with 1.5 µg of pMT2-HA-ERK-1
vector together with 1.5 µg of empty pRK-5 vector (lanes 1, 2, and
1320) or 1.5 µg of pRK-5-M2-FLAG-MLK3 (lanes
312) and 2 µg of either pCMV empty vector (lanes 5, 6,
13, and 14), pCMV-wild type c-Jun (lanes 7, 8, 15, and
16), pCMV-Jun-TAM-67 (tm, lanes 9, 10, 17, and 18),
or pCMV-Jun-DBM-3 (dbm, lanes 11, 12, 19, and 20).
A, ERK phosphorylation in HA-ERK immunoprecipitates (IP) was
determined by phospho-ERK immunoblotting (top panel), and HA-ERK
recovery was determined by ERK immunoblotting (bottom panel).
B, Jun phosphorylation at its activation sites was determined by
phospho-Jun immunoblotting of total cell lysates (top panel); Jun
expression was determined by Jun immunoblotting (middle panel), and
M2-FLAG-MLK3 expression was determined by M2-FLAG immunoblotting (bottom
panel). vec, vector.
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DISCUSSION
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Cellular response to environmental changes may be affected by variations in
cell type, development, and extracellular conditions. The mechanisms
underlying differential cellular responses are only partially understood and
can be explained in certain cases by differential gene and protein expression
patterns. The complex relationships between varying extracellular conditions
and cellular response to a distinct stimulus only recently began to be
elucidated at the mechanistic level, especially following the introduction of
high throughput technologies in genomics and proteomics. A key question that
remains unresolved is how a relatively small number of seemingly linear,
intracellular signaling pathways can mediate a large variety of responses to a
large variety of stimuli. Our results demonstrating a cross-talk relationship
between the stress-activated MLK-SEK-1-stress-activated protein kinase/JNK-Jun
pathway and the growth factor/MAPK/ERK pathway
(Fig. 6) offer one more answer
to this key question. Our results suggest that cells exposed to factors that
induce sustained activation of MLK3 and the JNK-Jun pathway will be less
responsive to growth factor-induced ERK activation. The recent finding that
TNF-
and ceramide activate MLK3
(17), together with the
results presented in this paper, suggest that prolonged exposure to
TNF-
or to other factors that result in ceramide generation, may render
cells less responsive to growth factor-induced ERK activation. This in turn
can result in altered cell cycle control, differentiation or cell growth, all
cellular responses involving the ERK/MAPK pathway.

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FIG. 6. A model describing the dual potential of MLK3 in regulating the ERK/MAPK
pathway. Sustained activation of MLK3 negatively regulates the ERK/MAPK
pathway by uncoupling ERK activation from MEK via activation of Jun
transcriptional activity. In addition, MLK3 has the potential to positively
regulate the ERK/MAPK pathway by directly phosphorylating and activating
MEK.
|
|
Our results demonstrating that MLK3 phosphorylates and activates MEK
suggest that under certain physiological conditions MLK3 may function as an
activator of the ERK/MAPK pathway, serving as an alternative path to the
Raf-mediated pathway (Fig. 6).
In addition, these findings imply that short and long phase MLK3 activation
may result in a different cellular response; whereas a short phase activation
may lead to ERK activation, a long phase activation results in an opposite
response. Also, although ERK activation may be mediated directly by MLK3, ERK
inhibition is mediated indirectly and involves the ability of MLK3 to activate
the JNK-Jun pathway and requires Jun-mediated gene transcription. These
implications point to a mechanism in which treatment with the same agonist may
result in a different cellular outcome, depending on the duration of the
treatment.
The MLK family of serine/threonine kinases consists of more than nine
related members that may have isoform-specific functions
(5). Knowledge of their
physiological function comes mainly from biochemical studies that classify
family members as MAPKKK in the stress-activated JNK pathway and from recent
genetic studies in Drosophila, which confirm the biochemical studies
and position the Drosophila single MLK isoform (d-MLK/slipper)
upstream of JNK (16,
17). The biological function
of MLKs in mammalian systems may be, however, more complicated than the one
identified in Drosophila. For example, in mammals, MLK isoforms are
differentially expressed in different tissues and have some variability in
their substrate specificity. In addition, differences in association with
scaffold proteins have been reported
(5). Our findings that MLK3 can
activate both SEK-1 and MEK-1 make this picture even more complex. For
example, hematopoietic cells that express high levels of MLK3 may respond to
cytokines that induce ceramide formation, such as TNF-
, by ERK
activation. On the other hand, cells that express little or no MLK3 would be
unable to activate ERK under the same conditions. In addition, TNF-
may
have a different effect on the same hematopoietic cell, depending on the
duration of exposure.
Our finding that the MLK3-induced attenuation of ERK activation depends on
c-Jun-mediated gene transcription is in agreement with recent findings showing
that stable cell lines expressing the constitutively active form of Jun
(v-Jun) exhibit attenuated ERK activation
(32). In another model,
activation of p38 by arsenite attenuates MEK activation by activating protein
phosphatase 1 and 2A (33).
The ability of MLK3 to activate the ERK/MAPK pathway and to induce a
transformed phenotype in NIH 3T3 cells has also been reported
(21). This work suggested that
this transformation is MEK-dependent; however, the role of JNK activation was
not examined. Thus, it is possible that the transformed phenotype was a result
of Jun activation, but the activity of the MEK-ERK pathway is required for
cell survival and normal growth.
An important question that remains unanswered is: What mediates the
uncoupling of ERK from MEK? Obvious candidates are phosphatases, which can
negatively regulate ERK activation. However, we were unable to detect elevated
expression of MKP13 (potential ERK specific phosphatases) in cells
expressing active MLK3 (data not shown). More detailed study will be required
to determine whether elevated expression or activity of phosphatases underlies
the observed MEK-ERK uncoupling.
An additional key question remaining to be resolved is: What is the
physiological role of the ability of MLK3 to activate MEK? This question can
be addressed by developing MLK forms differentially impaired in their ability
to activate MEK and SEK or by examining the effects of Raf- and MLK-specific
inhibitors on ERK activation under various physiological conditions in
different cell types.
In summary, the results in this paper propose a new role for MLK family
members in the regulation of the ERK/MAPK pathway both negative and positive.
These two opposite effects are mediated by different functions of MLK3: 1)
positive regulation through MLK3-mediated phosphorylation and activation of
MEK, and (2) negative
regulation through MLK3-induced activation of the SEK-1-JNK-Jun pathway that
requires Jun-mediated gene transcription. The physiological consequences of
the dual MLK3 potential remain to be uncovered.
 |
FOOTNOTES
|
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* This work was supported in part by National Institutes of Health Grants R01
GM 067134 (to G. T.) and R01 GM 558385 (to A. R.). The costs of publication of
this article were defrayed in part by the payment of page charges. This
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: Cardiovascular Research Institute,
Texas A&M University, HSC, 1901 S. 1st St., Bldg. 162, Temple, TX 76504.
Tel.: 254-778-4811 (ext. 1327); Fax: 254-899-6165; E-mail:
tzivion{at}medicine.tamu.edu.
1 The abbreviations used are: MLKs, mixed lineage kinases; JNK, c-Jun
amino-terminal kinase; MAPK, mitogen-activated protein kinase; ERK,
extracellular signal-regulated kinase; TNF, tumor necrosis factor; MEK,
MAPK/ERK kinase; PVDF, polyvinylidene difluoride; HA, hemagglutinin; GST,
glutathione S-transferase; MAPKKKs, mitogen-activated protein kinase
kinase kinases; MBP, myelin basic protein; EGF, epidermal growth factor; PMA,
phorbol 12-myristate 13-acetate. 
 |
ACKNOWLEDGMENTS
|
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We thank Joseph Avruch and Kenneth M. Baker for helpful discussions.
 |
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