Differential Activation of Two JNK Activators, MKK7 and SEK1, by MKN28-derived Nonreceptor Serine/Threonine Kinase/Mixed Lineage Kinase 2*

Syu-ichi HiraiDagger §, Kumi NodaDagger , Tetsuo Moriguchi, Eisuke Nishida, Akio YamashitaDagger , Tetsuya DeyamaDagger , Keiko FukuyamaDagger , and Shigeo OhnoDagger §

From the Dagger  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

    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.

    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 alpha , 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.

    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 SRalpha 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 beta -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 beta -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 beta -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 beta -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 beta -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 [gamma -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.

For the assay of recombinant JNK activators, 0.5 µg of GST-SEK1 and GST-MKK7 were used instead of the COS-1 cell lysate column fractions. The amounts of MalE-MST/10-757, GST-MEKK1DN, or other MalE-MST deletion mutants used in the assays are indicated in the figure legends.

To check the intrinsic kinase activities of MalE-MST/10-757 and GST-MEKK1DN, 1 µg of each protein was incubated in the 20 µl of assay buffer containing 5 µCi of [gamma -32P]ATP at 30 °C for 60 min. Phosphorylated proteins were detected by SDS-PAGE followed by autoradiography.

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).

    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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Fig. 1.   Identification of MST- and MEKK1-dependent JNK activators in COS-1 cell lysates. COS-1 cell lysates were fractionated by ion-exchange chromatography, and each fraction was analyzed for MST- and MEKK1-dependent JNK-activating activity by an in vitro reconstitution assay. A, schematic drawing of the strategy of fractionation. B, JNK-activating activity measured with HiTrap Q (Q Sepharose column) fractions. Aliquots of each fraction (2 µl) were subjected to assay with 3 µg of MalE-MST/10-757 (MST), 1.5 µg of GST-MEKKDN (MEKK), or 3 µg of MalE protein (control). The isolated points on the left indicate the activity of the total cell lysate loaded on the column (Total) and the run-through fraction (RT). C, JNK-activating activity measured with 1 µl of each Mono S column fraction. Isolated points on the left indicate the activity found in the HiTrap Q run through fraction loaded on the Mono S column (Ori) and the run-through fraction from the Mono S column (RT). D, Western blot analysis of the HiTrap Q fractions using anti-XMEK-2 antibody to detect SEK1 (upper panels) or anti-MKK7 antibody (lower panels). E, Western blot analysis of the Mono S fractions.

In addition to SEK1, MKK7 has been reported as a new MAPKK family member that activates JNK (18-22). To test whether the JNK activators found in the fractionated COS-1 cell lysates correspond to SEK1 and MKK7, proteins in each fraction were analyzed by Western blot using anti-XMEK-2, a frog homologue of SEK1, or anti-MKK7 antibody. As shown in the upper panel of Fig. 1D, SEK1 was found in the Q Sepharose fractions where the MST- or MEKK1-dependent JNK-activating activities were found. On the other hand, MKK7 was found in the Mono S fractions containing the MST- or MEKK1-dependent JNK-activating activities represented as the first higher peak (Fig. 1E, lower panel). Smaller amounts of MKK7 were also detected in the Mono S fractions corresponding to the latter lower peaks of MST- or MEKK1-dependent JNK-activating activity. The reason that MKK7 eluted at two different positions is not clear. Variants might be formed by posttranscriptional or posttranslational modification or by association with other proteins. Alternatively, the two peaks could correspond to two different gene products, both of which react with the anti-MKK7 antibody. In any case, both are relatively good substrates for MST compared with the Q Sepharose-bound JNK activator, SEK1.

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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Fig. 2.   The activation of recombinant SEK1 and MKK7 by MST or MEKK1 in vitro. Recombinant proteins fused with MalE or GST were produced in bacteria and used for the in vitro reconstitution assay. A, SDS-PAGE analysis of 3 µg of each recombinant protein (left panel). Asterisks indicate the position at which each protein is expected based on molecular mass. The arrowhead indicates the position of a co-purified ~70-kDa bacterial protein. The intrinsic kinase activity of MalE-MST/10-757 and GST-MEKK1DN was tested by incubating 1 µg each of protein with [gamma -32P]ATP. Phosphorylated proteins were detected by SDS-PAGE followed by autoradiography (right panel). B, GST-JNK1 was first incubated with 0.5 µg of GST-SEK1 or GST-MKK7 and 0-1.0 µg of MalE-MST/10-757 or GST-MEKK1DN in the presence of cold ATP. Then, recombinant c-Jun and [gamma -32P]ATP were added to measure JNK activity. Phosphorylated proteins were detected by SDS-PAGE followed by autoradiography. The amount of 32P incorporated into c-Jun was quantified and is indicated as relative amount at the bottom of the upper panel. c-Jun phosphorylation observed in the absence of SEK1 and MKK7 (GST) or JNK1 (-GST-JNK) are shown in the lower panel.

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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Fig. 3.   The activation of JNK in COS-1 cells by MST deletion mutants. COS-1 cells were cotransfected with the expression vector for His/T7-tagged JNK together with expression vectors for His/T7-tagged MST mutants. A, structure of the MST mutants. Numbers appearing in the name of each mutant indicate the part of MST (953 amino acids in all) they contained. Amino acid residues 140-185, corresponding to kinase subdomains III-V, were deleted in His/T7-MST/KN. B, the expression of His/T7 tagged protein was analyzed by Western blotting using anti-T7 tag antibodies (upper panel). His/T7-JNK1 appeared as a 50-kDa protein. Asterisks indicate the positions of the MST mutants. The activity of His/T7-tagged JNK1 was measured by in gel kinase assay using GST-CREBP1/1-143 as a substrate (lower panel). The values shown at the bottom are relative amounts of 32P incorporated into the substrate.

Next, we tested whether deletion of nonkinase domains would affect the substrate specificity of MST for JNK activators found in COS-1 cell lysates. MST deletion mutants were produced in bacteria as fusion proteins with a maltose-binding protein (Fig. 4, A and B) and used in the in vitro reconstitution experiment. As shown in Fig. 4C, all MalE-fused deletion mutants, including the naked kinase domain, showed very similar substrate specificities; i.e. they were more effective activators of JNK activators in the Mono S-bound fractions than in the Q Sepharose-bound fractions. Substrate specificity was also tested using recombinant GST-SEK1 and GST-MKK7 (Fig. 4D). Again, the deletion of regions outside the kinase domain did not affect the substrate specificity of MST. With respect to the GST-SEK1-dependent JNK-activating activity, all MalE-MST mutants showed only 13-20% of the activity observed for GST-MEKK1DN, whereas the values rose to 75-80% for the GST-MKK7 dependent JNK-activating activity.


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Fig. 4.   Activation of JNK activators by MST deletion mutants in vitro. A, structures of MST deletion mutants produced in bacteria. B, MalE-fused MST mutant proteins (3 µg each) or MST/10-444 (1 µg) used for the assay were analyzed by SDS-PAGE. The MST/10-444 protein was obtained by treating the MalE-MST/10-444 fusion protein with factor Xa, and the released maltose-binding protein (MalE) was removed with amylose resin. The arrowhead indicates the position of residual MalE protein remaining in the MST/10-444 fraction. Its size is smaller than MalE protein produced with control pMal-c2 vector (left lane), which is flanked by an extra C-terminal tail originating from the linker and pMal-c2 vector sequence. C, JNK-activating activities in COS-1 cell lysates fractionated with HiTrap Q and Mono S columns were measured as described in the legend to Fig. 1 in the presence of 1.1 µM (2 to ~3 µg) MalE-MST deletion mutants. D, the activation of GST-SEK1 and GST-MKK7 by MST deletion mutants. The assay was carried out as described in Fig. 2 in the presence of 1.1 µM (2 to ~3 µg) of MalE-MST deletion mutants. A limited amount (250 nM) of MST/10-444 was used for the assay (asterisk). The JNK-activating activity of GST-SEK1 or GST-MKK7 induced by each mutant is expressed as a percentage of the activity induced by 1 µg of GST-MEKK1DN.

To eliminate the possibility that the fused MalE protein is responsible for the more effective activation of MKK7 by the recombinant MalE-MST fusion proteins, MST/10-444 was released from MalE-MST/10-444 fusion protein by using a site-specific protease, factor Xa, and used for the in vitro reconstruction experiment. As shown in Fig. 4D, the substrate specificity of MST/10-444 is very similar to that of the MalE-MST/10-444 fusion protein. Therefore, fusion to MalE is not responsible for the substrate specificity of MST.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha , 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 alpha  (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.

    ACKNOWLEDGEMENTS

We thank Drs. Toshio Maekawa and Shunsuke Ishii for the gift of the CREBP-1 cDNA clone.

    FOOTNOTES

* 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.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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