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J. Biol. Chem., Vol. 278, Issue 29, 26715-26721, July 18, 2003
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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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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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
(18–20)
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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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 Transfection—COS-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 Purification—Cells 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 Assays—Raf 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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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 Vivo—To 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 Activation—Because 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 Activation—To 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 7–11; 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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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 7–9) 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 Activation—To 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 (23–25), 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 3–6 with lanes 9–12). 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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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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 MKP1–3 (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.
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FOOTNOTES |
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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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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