From the Department of Molecular Biology, Yokohama
City University School of Medicine, 3-9 Fukuura, Kanazawa-ku,
Yokohama 236, Japan and the ¶ Department of Biophysics, Graduate
School of Science, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan
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ABSTRACT |
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MKN28-derived nonreceptor type of serine/threonine kinase/mixed lineage kinase 2 (MST/MLK2) directly phosphorylates and activates SEK1/MKK4/JNKK1/SKK1 in vitro, thereby acting as a mitogen-activated protein (MAP) kinase kinase kinase in the JNK/SAPK pathway (Hirai, S.-i., Katoh, M., Terada, M., Kyriakis, J. M., Zon, L. I., Rana, A., Avruch, J., and Ohno, S. (1997) J. Biol. Chem. 272, 15167-15173). The in vitro reconstitution system for the kinase cascade allowed us now to identify JNK/SAPK activators involved in the MST/MLK2-dependent activation of JNK/SAPK in vivo. We show that at least two distinct MST/MLK2-dependent JNK/SAPK activators are present in the fractionated COS-1 cell lysate, and that they appear to be SEK1/MKK4/JNKK1/SKK1 and MKK7/JNKK2/SKK4 by Western blot analysis. Notably, a majority of the MST/MLK2-dependent JNK/SAPK-activating activity is found in MKK7-containing fractions, whereas the MEKK1-dependent activity is comparably distributed in SEK1- and MKK7-containing fractions. Moreover, MST/MLK2 activates recombinant MKK7 more effectively than recombinant SEK1, whereas MEKK1 activates both to a similar extent. In addition, the deletion analysis on MST/MLK2 showed that the kinase domain is responsible for the determination of substrate specificity. These results provide a molecular aspect to the differential regulation of the two JNK activators by a variety of cellular stimuli.
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INTRODUCTION |
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JNK/SAPK is a
MAPK1-related protein kinase
mainly involved in cellular responses against cytokines, such as tumor
necrosis factor , Fas-ligand, and interleukin-1, and stress-inducing
agents, such as ultraviolet light, osmotic pressure, and heat (1-3). The diversity of the JNK/SAPK-inducing extracellular stimuli indicates the presence of multiple pathways to activate this protein kinase. In
fact, there have been an increasing number of MAPKKK class protein
kinases acting in the JNK/SAPK pathway now being reported, and these
might be involved in the activation of JNK/SAPK by distinct extracellular stimuli (4-7). We and others have reported that members
of the mixed lineage kinase (MLK) family, MUK/DLK/ZPK, SPRK/PTK1/MLK3,
and MST/MLK2, activate the JNK/SAPK pathway (8-12). Furthermore, we
have shown that MST/MLK2 can directly phosphorylate and activate SEK1,
a MAPKK class protein kinase that activates JNK/SAPK (12). Therefore,
MST/MLK2, and probably other members of the MLK family as well, acts as
a MAPKKK in the JNK/SAPK pathway.
A direct activator of JNK/SAPK, the MAPKK class protein kinase SEK1/MKK4/JNKK1/SKK1 has been cloned, and its involvement in the activation of JNK/SAPK by MEKK1 has been demonstrated (13-15). On the other hand, two JNK/SAPK activators have been identified in the column fractions of rat 3Y1 cell extracts and three in human KB cell extracts (16, 17). One of these activators corresponds to SEK1, but the others appear to be distinct new activators the primary structures of which are not known. More recently, a second JNK/SAPK activator, MKK7/JNKK2/SKK4, has been cloned (18-22). Intensive studies to identify the MKK7 substrate have demonstrated that MKK7 selectively activates JNK/SAPK and has very little ability to activate other MAP kinases, extracellular signal-regulated kinases, or p38 MAP kinases (18-22). On the other hand, little is known about the MAPKKKs that act on MKK7.
MLKs have been shown to comprise a new family of protein kinases that share a characteristic protein kinase domain that shows structural features of both tyrosine-specific and serine/threonine-specific protein kinases and two leucine-zipper-like motifs located proximal to the C-terminal end of the protein kinase domain (23). The functional significance of these motifs is still unclear; however, the physical interaction of a small GTP-binding protein, Cdc42, and a hematopoietic progenitor kinase, HPK1, with SPRK/PTK1/MLK3 in vitro has been reported (24, 25). Both Cdc42 and HPK1 could be involved in the regulation of SPRK/PTK1/MLK3 or other members of the MLK family. SEK1 has been reported to be a substrate of MLKs. MLKs activate epitope-tagged SEK1 in cultured cells and phosphorylate SEK1 in vitro (9, 11, 12). However, no MAPKKs relevant to the activation of JNK by MLKs in cells have yet been identified.
We developed a unique system to identify the JNK/SAPK activators activated by MST/MLK2. This system involves an in vitro reconstitution assay of the kinase cascade using recombinant MST/MLK2 and JNK1 and the JNK/SAPK activators to be tested. By subjecting a fractionated COS-1 cell lysate to this system, we found at least two MST-dependent JNK/SAPK activators. Furthermore, using recombinant SEK1 and MKK7 proteins, we found that MST/MLK2 activates MKK7 more effectively than SEK1, whereas MEKK1 activates both to a similar extent.
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EXPERIMENTAL PROCEDURES |
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Construction of Plasmids and Preparation of Recombinant
Proteins--
To construct a bacterial expression vector for MEKK1
fused to GST, a cDNA encoding the C-terminal 687 amino acids of
mouse MEKK1 (MEKK1DN) (8) was cloned into pGEX-2T (Amersham Pharmacia Biotech) vector. A bacterial expression vector for GST-CREBP1/1-143, which served as a substrate in gel kinase assay, was constructed with
pGEX-3X vector (Amersham Pharmacia Biotech) and a cDNA encoding the
N-terminal 143 amino acids of CREBP1/ATF2. GST-MKK7 was constructed with pGEX-2T vector (Amersham Pharmacia Biotech) and a cDNA
encoding mouse MKK7 (22). Bacterial expression vectors for MST deletion mutants fused to MalE were constructed with pMal-c2 (New England Biolabs) vector and cDNAs encoding different parts of MST as
indicated in Fig. 4. These cDNAs were also cloned into a mammalian
expression vector containing the SR promoter and an N-terminal
His/T7-tag sequence. Mammalian expression vectors for MST and JNK1 and
bacterial expression vectors for MalE-MST/10-757, GST-SEK1, and
GST-JNK1 have been described elsewhere (12). GST and MalE fusion
proteins were produced in Escherichia coli (DH5) and
purified by standard protocols on glutathione-Sepharose and amylose
resin, respectively. To obtain MalE-free MST/10-444, 500 µg of MalE
fusion protein was mixed with 5 µg of factor Xa (Sigma) in 500 µl
of 150 mM NaCl, 2.5 mM CaCl2, 5 mM
-mercaptoethanol, 10 mM Tris-HCl, pH 7.5, and incubated at 25 °C for 2.5 h. Then, excised MalE protein
and uncut MalE fusion protein were removed by passage through a 1-ml amylose resin column equilibrated with 150 mM NaCl, 5 mM
-mercaptoethanol, 10 mM Tris-HCl, pH 7.5. Recombinant c-Jun was produced in bacteria using a PET8c expression
vector (8). Protein solutions were dialyzed against 100 mM
NaCl, 5 mM
-mercaptoethanol, 10% glycerol, 10 mM Tris-HCl, pH 7.5, and stored at
80 °C until
use.
Fractionation of COS-1 Cell Lysate--
COS-1 cells were
routinely cultured in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum. Cells grown in ten 10-cm dishes (~5 × 107 cells) were washed three times with
phosphate-buffered saline and harvested. The cell pellet was
resuspended in 5 ml of TG buffer (20 mM Tris-HCl, pH 7.5, 25 mM -glycerophosphate, 2 mM EGTA, 2 mM dithiothreitol, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.1% Triton
X-100, 10% glycerol). The cells were disrupted in a Dounce homogenizer
by 40 strokes with a tight pestle and left to stand on ice for 30 min.
The cell lysate was centrifuged at 100,000 × g for 30 min, and the supernatant was loaded onto a 1-ml Q Sepharose column (HiTrap Q, Amersham Pharmacia Biotech) equilibrated with TG buffer. The
protein bound to the column was eluted with a 10-ml linear gradient of
0-0.5 M NaCl in TG buffer, and 1-ml fractions were collected. The unadsorbed fractions from the Q Sepharose column were
loaded onto a 0.1-ml Mono S column (Amersham Pharmacia Biothech) equilibrated with TG buffer. The protein bound to the column was eluted
with a 2-ml linear gradient of 0-0.5 M NaCl, and 0.1-ml fractions were collected.
Assay for JNK Activators: in Vitro Reconstruction of the Kinase
Cascade--
For the assay of JNK activators in COS-1 cell lysates, 2 µl from each of the Q Sepharose column fractions or 1 µl from each of the Mono S column fractions was added to 20 µl of ice-cold assay
buffer (15 mM HEPES, pH 7.5, 12.5 mM
MgCl2, 12.5 mM -glycerophosphate, 12.5 mM p-nitrophenyl phosphate, 0.1 mM
Na3VO4, 1 mM dithiothreitol, 20 µg/ml maltose-binding protein, 30 mM maltose, 100 µM ATP) containing 0.2 µg (140 nM) of
GST-JNK1 and 3 µg (~1.1 µM) of MalE-MST/10-757 or
1.5 µg (~700 nM) of GST-MEKK1DN and incubated at
30 °C for 60 min. Then, 2 µl of a substrate mix containing 25 µM ATP, 2 µCi of [
-32P]ATP, and 1 µg
of recombinant c-Jun was added to each tube, and the samples were
further incubated for 15 min at 30 °C. The reactions were stopped by
adding SDS-PAGE sample buffer, and the phosphorylation of c-Jun was
detected by SDS-PAGE followed by autoradiography. The amount of
32P incorporated was quantified with a Fuji BAS2000 image
analyzer.
Transfection of COS-1 Cells and in Gel Kinase Assay-- COS-1 cells were transfected by an electroporation method using 16 µg of DNA for 5 × 106 cells seeded in four 10-cm dishes. After transfection, the cells were cultured for an additional 48 h before harvest. Cells were lysed in SDS-PAGE sample buffer, and the activity of the His/T7-tag JNK1 expressed in the cells was measured by in gel kinase assay (8) using GST-CREBP1/1-143 as a substrate.
Antibodies-- SEK1 was detected by Western blotting using a rabbit antibody against recombinant XMEK2, a Xenopus homologue of human SEK1 (16). MKK7 was detected using a rabbit antibody against recombinant mouse MKK7 (22). His/T7-tagged protein was detected using an anti T7-tag monoclonal antibody (Novagen). A peroxidase-linked antibody against rabbit or mouse IgG was used as a secondary antibody and detected by an ECL detection system (Amersham Pharmacia Biotech).
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RESULTS |
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COS-1 Cells Contain at Least Two MST/MLK2-dependent JNK/SAPK Activators-- We have reported that the overexpression of MST in COS-1 cells causes the activation of JNK1 (Ref. 12; see also Fig. 3). Even though MST directly phosphorylates and activates SEK1 (12), other substrates of MST could be acting in COS-1 cells to activate JNK. To identify any MST-dependent JNK activator(s) in COS-1 cells, we fractionated COS-1 cell lysates and assayed each fraction for MST-dependent JNK activator activity with an in vitro reconstitution assay system. The assay mixture contained recombinant GST-JNK1 and its substrate c-Jun, and the JNK-activating activity in the fractionated cell lysates was estimated by the ability of GST-JNK1 to phosphorylate c-Jun. COS-1 cell lysates were first chromatographed on Q Sepharose, and the unbound fraction was chromatographed on Mono S (Fig. 1A). In the absence of MST, a single weak peak of JNK-activating activity was observed in the Mono S fractions (Fig. 1C). The addition of a recombinant MST, MalE-MST/10-757, to the assay system induced the appearance of three peaks, one in the Q Sepharose fractions (Fig. 1B) and the others in the Mono S fractions (Fig. 1C). One of peaks observed in the Mono S fractions was located at the same position as the weak peak observed in the absence of MST. These three peaks were also observed when a recombinant MEKK1, GST-MEKK1DN, was added instead of MST. Notably, the addition of MEKK1 produced a higher peak in the Q Sepharose binding fraction, whereas the height of the two peaks in the Mono S fractions was rather lower than those seen in the presence of MST (Fig. 1, B and C). The difference in the relative height of each peak in response to MST and MEKK1 reflects the different substrate specificity of these two MAPKKKs on different JNK activators present in each peak. The different substrate specificities of MST and MEKK1 are also shown by the observation that more than half of the MEKK1-dependent JNK-activating activity found in the total cell lysate bound to Q Sepharose, whereas a large part of the MST-dependent JNK-activating activity passed through the Q Sepharose column (see Fig. 1B, Total and RT).
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MST/MLK2 Activates Recombinant MKK7 More Efficiently than Recombinant SEK1-- To confirm that MST and MEKK1 activate MKK7 and to further verify the difference in the substrate specificities of MST and MEKK1, we used recombinant SEK1 and MKK7 (Fig. 2A) for the in vitro reconstitution assay system. The abilities of MST and MEKK1 to activate these MAPKKs were monitored by measuring their abilities to activate JNK1, the activity of which was in turn estimated by its ability to phosphorylate c-Jun. Both MalE-MST/10-757 and GST-MEKK1DN activate GST-SEK1 as previously reported. However, the ability of MalE-MST/10-757 to activate GST-SEK1 is much lower than that of GST-MEKK1DN; when 1 µg of MalE-MST/10-757 or GST-MEKK1 was used for the assay, only a 2.0-fold activation of the JNK-pathway was observed with MalE-MST/10-757, whereas a 14.9-fold activation was observed with GST-MEKK1DN (Fig. 2B). When the amount of MalE-MST/10-757 was increased to 4 µg, the observed activation was greater than 5-fold (12). However, this value is still smaller than that obtained with less GST-MEKK1DN. On the other hand, both of these MAPKKKs activate GST-MKK7 to a comparable extent: approximately 4-fold and 6-fold for MalE-MST/10-757 and GST-MEKK1DN, respectively (Fig. 2B). Although relatively more GST-MEKK1DN was degraded than MalE-MST/10-757, the kinase activity of GST-MEKK1DN was much higher than that of MalE-MST/10-757 when estimated by the level of autophosphorylation (Fig. 2A, right panel). Therefore, the greater ability of GST-MEKK1DN to activate GST-SEK1 may be explained in part by the higher intrinsic kinase activity of this recombinant protein. However, it is obvious that MalE-MST/10-757 and GST-MEKK1DN show different substrate specificities for GST-SEK1 and GST-MKK7 and that MalE-MST/10-757 activates GST-MKK7 more efficiently than GST-SEK1. Notably, no induction of JNK activity was observed when GST was used instead of GST-SEK1 or GST-MKK7, and no c-Jun phosphorylation was observed when GST was used instead of GST-JNK1 (Fig. 2B, lower panel). Therefore, all of the activities of MalE-MST/10-757 and GST-MEKK1DN discussed above depend highly on those MAPKK- and MAPK-class protein kinases.
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The Kinase Domain of MST/MLK2 Is Responsible for Substrate Specificity-- MST/MLK2 has an SH3 domain in its N-terminal portion and two leucine-zipper-like motifs and a putative Rac/Cdc42 binding motif proximal to the C-terminal end of its kinase domain. To test whether these motifs contribute to substrate specificity, we constructed MST deletion mutants lacking these motifs. First, the ability of these mutants (Fig. 3A) to induce JNK1 activity in COS-1 cells was tested by a cotransfection experiment followed by the in gel kinase assay to monitor the activity of the epitope-tagged JNK1 cotransfected with each MST mutant. As shown in Fig. 3B, lower panel, all mutants except MST/KN, the kinase domain of which was partly deleted, were able to activate JNK1 in COS-1 cells. Although all C-terminal deletion mutants showed higher activities than tag-MST/10-953 carrying an intact C-terminal domain, this higher activity can mostly be explained by the higher expression level of these mutants (Fig. 3B, upper panel). Therefore, the specific ability of MST to induce JNK1 in COS-1 cells is not significantly affected by the deletion of amino acids flanking its kinase domain.
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DISCUSSION |
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We identified two MST-dependent JNK activators in fractionated COS-1 cell lysates by an in vitro reconstitution assay system using recombinant MST and JNK1. Western blot analysis indicated that one of these activators corresponds to SEK1/MKK4/JNKK1/SKK1 and the other to the recently identified JNK activator MKK7/JNKK2/SKK4. The activation of these MAPKKs by MST/MLK2 was further verified by the assay using recombinant proteins. Notably, the ability of MST/MLK2 to activate MKK7 was much higher than its ability to activate SEK1. MKK7 selectively activates JNK, whereas other MAP kinases, such as extracellular signal-regulated kinases and p38-MAP kinases, are only partially activated (18-22). On the other hand, SEK1 activates p38 MAPK, as well as JNK (15, 21, 22). Therefore, the above findings are in line with our previous observation in vitro that MST is a rather selective activator of the JNK pathway (12).
The experiment using sek1/
ES cells verified
the involvement of SEK1 in the activation of JNK by anisomycin, heat
shock, and the overexpression of MEKK1 (26, 27). Furthermore, the
endogenous or overexpressed SEK1 in culture cell lines, such as HeLa,
KB, and NIH3T3 cells, is activated by anisomycin, osmotic shock, or the
overexpression of MEKK1 (17, 20-22). On the other hand, MKK7 is
activated by interleukin-1 and tumor necrosis factor
, which hardly
activate SEK1, as well as by anisomycin, osmotic shock, and the
overexpression of MEKK1, all of which also activate SEK1 (20-22).
Therefore, SEK1 and MKK7 must have different specific upstream
activators in addition to some common activators.
Our finding that MST is a more effective activator of MKK7 than SEK1
provides a clue to the molecular basis for the selective activation of
MKK7 by certain stimuli. The selective activation of MKK7 by
interleukin-1 or tumor necrosis factor (21, 22) suggests the
involvement of MST in the activation of MKK7 by some of these stimuli.
However, the signaling pathway by which MST activity is induced is not
clear. Notably, overexpressed MST acts as a potent JNK activator
without any stimulus, and the deletion of the kinase domain-flanking
region does not produce any significant changes in its ability to
activate MKK7 in vitro or in JNK activation in COS-1 cells
(Figs. 3 and 4). Therefore, the activity of MST might be regulated by
controlling the amount of protein in addition to posttranslational
modification.
The substrate specificity of MST depends on its kinase domain, but other typical structures in MST, including the SH3 domain, the leucine-zipper-like domain, and the Rac/Cdc42 binding motif, are not essential for the preferential activation of MKK7. The kinase domains of members of the MLK family, including MST, show higher homologies to those of TAK and Raf than to that of MEKK1. When MAPKKK class protein kinases are classified according to the amino acid sequences of their kinase domains, MLK family members and MEKK family members fall into different groups (12). Conceivably, the difference in the amino acid sequences of the MAPKKK kinase domains manifests itself as the different substrate specificities of MAPKKKs for two JNK activators, whereas the difference is not related to downstream specificity at the MAP kinase level. It would then be interesting to know whether JNK-activating MAPKKKs that belong to the same group as MST, such as MUK/DLK/ZPK, SPRK/PTK1/MLK3, and TAK, preferentially activate MKK7 as MST does.
Is there any selective activator of SEK1? It has been reported that the
activation of JNK by the ectopical expression of MEKK1 is severely
impaired in sek1/
ES cells. On the other
hand, ultra violet light or osmotic shock induces JNK activation in
sek1
/
ES cells as in
sek1+/+ ES cells (26, 27), and this may depend
on another MAPKK, MKK7. These observations indicate that MEKK1 is a
selective activator of SEK1, and this conclusion appears to be
inconsistent with the observation made by us and others that MEKK1
activates both SEK1 and MKK7 (21). This discrepancy can be explained by
the absence of MKK7 and the presence of an unidentified type of JNK
activator (22) in ES cells that is activated by ultraviolet light or
osmotic shock but not by MEKK1. Alternatively, the interaction of MKK7 with MEKK1 could be blocked by a more stable interaction of MKK7 with
other MAPKKKs in ES cells.
Despite the increasing information about the differences upstream of SEK1 and MKK7, little is known about the specific roles of these protein kinases in cellular responses. The rather ubiquitous distribution of both enzymes in mouse tissues and cultured cell lines (18, 20, 22) decreases the possibility that their functions are segregated in different cell types. Why then do two, and possibly more, MAPKKs act on JNK? The activity of each MAPKK might be regulated by a different set of MAPKKKs, as partially shown in this paper, which are activated by different sets of stimuli. Therefore, the presence of multiple MAPKKs increases the number of stimuli that can induce JNK activation. Moreover, SEK1 can activate MAPKs other than JNK, such as p38 MAPK (15, 21, 22), indicating the ability of SEK1 to induce specific cellular responses that cannot be induced by MKK7. From another point of view, each MAPKK might be localized in different places in cells by interacting with specific cellular proteins, as reported for SEK1, which interacts with actin-binding protein p280 (28). It is then conceivable that each MAPKK activates a different set of JNK molecules in a cell, and this may induce a specific cellular response. Therefore, MST might be involved in the regulation of a specific part of the cellular response that results from JNK activation. The identification of such cellular response(s), as well as the MST-activating mechanism, will verify the physiological significance of the MST-JNK signaling pathway.
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ACKNOWLEDGEMENTS |
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We thank Drs. Toshio Maekawa and Shunsuke Ishii for the gift of the CREBP-1 cDNA clone.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and by grants from the Japan Society for the Promotion of Science.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. Tel.: 81-45-787-2597; Fax: 81-45-785-4140; E-mail: sh3312{at}med.yokohama-cu.ac.jp.
1 The abbreviations used are: MAP, mitogen-activated protein; MAPK, MAP kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; MST, MKN28-derived nonreceptor type of serine/threonine kinase; GST, glutathione S-transferase; MalE, maltose-binding protein; PAGE, polyacrylamide gel electrophoresis; MLK, mixed lineage kinase.
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REFERENCES |
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