Murine Protein Serine/Threonine Kinase 38 Activates Apoptosis Signal-regulating Kinase 1 via Thr838 Phosphorylation*

Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H2O2 or tumor necrosis factor α treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr838 phosphorylation and enhanced ASK1-mediated signaling to both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H2O2-mediated apoptosis by enhancing the ASK1 activity through Thr838 phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.

Murine protein serine/threonine kinase 38 (MPK38), also known as maternal embryonic leucine zipper kinase (Melk), is a member of the AMP-activated protein kinase-related serine/ threonine kinase family (9,10). MPK38 was originally identified as a murine counterpart for its human homolog, HPK38/ hMelk/KIAA175, that may be involved in the proliferation of interleukin-4-induced normal human keratinocytes (9). The importance of MPK38 in oncogenesis is also underscored by the finding that MPK38 expression is increased in tumor-derived progenitor cells as well as in cancers of nondifferentiated cells (11)(12)(13). However, the physiological regulation and functions of MPK38 have remained unclear.
In Vivo and in Vitro Binding Assay-Each plasmid DNA utilized in the study was transfected into HEK293, 293T, or HaCaT cells using WelFect-Ex TM Plus (WelGENE, Daegu, Korea), according to the manufacturer's instructions. In vivo binding assays were performed as described previously (18). For native PAGE to determine the in vitro binding between MPK38 and ASK1, the procedure was the same as that of denaturing SDS-PAGE, except that solutions did not contain SDS or ␤-mercaptoethanol, and samples were not boiled prior to loading (18).
In Vitro Kinase Assay-In vitro kinase assays were performed as described previously (6). Cells transiently transfected with the indicated expression vectors were harvested and lysed with buffer (20 mM HEPES, pH 7.9, 10 mM EDTA, 0.1 M KCl, and 0.3 M NaCl). The cleared lysates were subjected to immunoprecipitation by incubation for 2 h at 4°C with the appropriate antibodies. After washing the immunoprecipitate three times with lysis buffer, then twice with each kinase buffer (for ASK1, 20 mM Tris-HCl, pH 7.5, 0.1 mM sodium orthovanadate, 1 mM DTT, and 20 mM MgCl 2 ; for MKK3/6, 25 mM HEPES, pH 7.4, 0.1 mM sodium orthovanadate, 25 mM sodium ␤-glycerophosphate, 2 mM DTT, and 25 mM MgCl 2 ; for p38, 50 mM HEPES, pH 7.4, 1 mM sodium orthovanadate, 0.2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 1 mM MgCl 2 ), the immunoprecipitates were assayed for the indicated protein kinase activities in the presence of each kinase buffer containing 5 g of recombinant GST-tagged substrates. The reaction mixtures were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and detected by autoradiography. Recombinant GST-tagged ZPR9, MKK6(K82A), p38, and ATF2 were used as substrates for MPK38, ASK1, MKK3 or MKK6, and p38 mitogen-activated protein kinase (MAPK), respectively. Protein concentration was determined by the Bradford assay.
Assays for MPK38 and JNK Activities-Cells were transiently transfected with GST-tagged MPK38 or JNK, along with indicated expression vectors, and solubilized with lysis buffer (20 mM HEPES, pH 7.9, 10 mM EDTA, 0.1 M KCl, and 0.3 M NaCl). The cleared lysates were precipitated by glutathione-Sepharose beads. The GST precipitates were washed three times with lysis buffer and twice with kinase buffer (for MPK38, 50 mM HEPES, pH 7.4, 1 mM DTT, and 10 mM MgCl 2 ; for JNK, 20 mM HEPES, pH 7.6, 0.1 mM sodium orthovanadate, 25 mM sodium ␤-glycerophosphate, 2 mM DTT, and 20 mM MgCl 2 ), and then subjected to an in vitro kinase assay using recombinant ZPR9 (19) or c-Jun as a substrate in the presence of 5 Ci of [␥-32 P]ATP, followed by SDS-PAGE and autoradiography.
Small Interfering RNA (siRNA) Experiments-The MPK38 siRNAs of the oligonucleotide 1 (5Ј-CAGGCAGACAAUGGA-GGAUTT-3Ј) targeting a coding region (amino acids 297-303) and oligonucleotide 2 (5Ј-AACCCAAGGGUAACAAGGATT-3Ј) targeting a coding region (amino acids 156 -162) on MPK38 (GenBank TM accession number NM010790), as well as ASK1 siRNA (5Ј-GGUAUACAUGAGUGGAAUUTT-3Ј) (20), were synthesized from SamChully Pharm. Co., Ltd. (Seoul, Korea). The sense and antisense oligonucleotides for each siRNA were mixed and heated at 90°C for 2 min, and the combined reaction was incubated at 30°C for 1 h. HEK293 cells grown were plated in 6-well flat-bottomed microplates (Nunc) at a concentration of 2 ϫ 10 5 cells per well the day before transfection. siRNA oligonucleotides with the indicated concentrations were transfected into cells using WelFect-Ex TM Plus. After 48 h of transfection, immunoblottings were carried out to confirm the down-regulation of target proteins.
Luciferase Reporter Assay-293T cells were transfected according to the WelFect-Ex TM Plus method with the AP-1-Luc reporter plasmid, along with each expression vector as indicated. After 48 h, the cells were harvested, and luciferase activity was monitored with a luciferase assay kit (Promega) following the manufacturer's instructions. Light emission was determined with a Berthold luminometer (Microlumat LB96P). Total DNA concentration was kept constant by supplementing with empty vector DNAs. The values were adjusted with respect to expression levels of a cotransfected ␤-galactosidase reporter control, and experiments were repeated at least three times.
Assays for Apoptosis-For cell death experiments using the green fluorescent protein (GFP) system (18), HEK293 cells grown on sterile coverslips were transfected with pEGFP (a GFP-encoding expression vector) together with expression vectors as indicated. After 24 h of transfection, the cells were washed with phosphate-buffered saline and then incubated for 40 h in serum-free medium. The cells were fixed with ice-cold 100% methanol, washed three times with phosphate-buffered saline, and then stained with 4Ј,6Ј-diamidino-2-phenylindole dihydrochloride. The 4Ј,6Ј-diamidino-2-phenylindole dihydrochloride-stained nuclei of GFP-positive cells were analyzed for apoptotic morphology by fluorescence microscopy. The percentage of apoptotic cells was calculated as the number of GFP-positive cells with apoptotic nuclei divided by the total number of GFP-positive cells. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed according to the manufacturer's instructions (Roche Applied Science). Cells exposed to 1 mM H 2 O 2 for 9 h were used as a positive control.

Direct Interaction of MPK38 and ASK1 in Vivo and in Vitro-
We initially performed in vitro kinase assays using 293T cells to identify MPK38-specific stimuli that modulate MPK38 kinase activity. Among the various stimuli tested, MPK38 precipitate from cell lysates treated with H 2 O 2 at concentrations of more than 0.5 mM significantly induced MPK38 kinase activity compared with untreated MPK38 precipitate (Fig. 1A, left). We next analyzed the kinetics of MPK38 and ASK1 kinase activity in H 2 O 2 -stimulated 293T cells. The stimulation of MPK38 and ASK1 kinase activity was detected at 5 min after treatment with 2 mM H 2 O 2 , peaked at 30 min, and decreased thereafter (Fig.  1A, right). These results suggested that cross-talk between MPK38 and ASK1 signaling pathways may exist as H 2 O 2 is well known to activate ASK1.
To test whether MPK38 is associated with ASK1 in cells, we performed cotransfection experiments using GST-MPK38 and FLAG-ASK1 expression vectors. The interactions between FLAG-tagged ASK1 proteins and GST-MPK38 fusion proteins were analyzed by immunoblotting with an anti-FLAG antibody. ASK1 was detected in the coprecipitate only when coexpressed with GST-MPK38 but not with control GST alone, demonstrating that MPK38 physically interacts with ASK1 ( Fig. 1B,  left). To confirm the endogenous interaction between MPK38 and ASK1, we carried out coimmunoprecipitation experiments using endogenous MPK38 and ASK1 in HEK293 cells. Immunoprecipitation of endogenous MPK38 by anti-MPK38 antibody and then immunoblotting with an anti-ASK1 antibody led to the clear identification of an interaction between the two endogenous MPK38 and ASK1 proteins (Fig. 1B, middle). We have further determined this association using other cell lines, including NIH 3T3 cells and R1.1 hematopoietic cells highly expressing MPK38 (15), and we confirmed that this association could occur in vivo (data not shown). We also analyzed the in vitro association of purified, recombinant ASK1 with MPK38 using nondenaturing PAGE. Autophosphorylated recombinant MPK38 was incubated with an unlabeled, recombinant kinase-dead form of ASK1 (ASK1(K709R)) or with GST as a nonspecific control. A shift in the mobility of 32 P-labeled MPK38 was clearly evident upon incubation in the presence of ASK1(K709R), but it was undetectable when 32 P-labeled MPK38 was incubated with GST alone or in the absence of ASK1(K709R), providing additional evidence of a physical association between MPK38 and ASK1 (Fig. 1B, right).
To investigate whether the activity of both kinases was involved in the association between MPK38 and ASK1, we used an in vivo binding assay to examine the effects of the kinasedead forms of MPK38 and ASK1 on MPK38-ASK1 complex formation. Expression of kinase-dead ASK1 (ASK1(K709R)) or ASK1(T838A), which is defective in ASK1 activation, resulted in a significant reduction of complex formation compared with expression of wild-type ASK1 (Fig. 1C, left and middle). Similar results were also observed with the kinase-dead MPK38 (MPK38(K40R)) in the analysis of the association between MPK38 and ASK1 (Fig. 1C, right). Consistent with this, the interaction was very weakly detected when the kinase-dead forms of both MPK38 and ASK1 were expressed (Fig. 1C, right, 6th lane). In addition, H 2 O 2 treatment did not contribute to the modulation of the association between MPK38 and ASK1 containing kinase-dead MPK38 and/or ASK1 (Fig. 1D, 3rd and 4th lanes versus 5th to 10th lanes), indicating that the kinase activity of both MPK38 and ASK1 is important for Modulation of MPK38-ASK1 Complex Formation by ASK1 Stimuli-We assessed whether H 2 O 2 , a stimulator of ASK1, can influence the MPK38-ASK1 complex formation in HEK293 cells transfected with plasmid vectors expressing GST-MPK38 and FLAG-ASK1. After 48 h of transfection, cells were incubated in media with or without 2 mM H 2 O 2 for 30 min. MPK38 was precipitated and the coprecipitation of ASK1 was determined by anti-FLAG immunoblot. MPK38-ASK1 association was significantly increased by H 2 O 2 treatment compared with untreated control (Fig.  1E, upper left). Similarly, the interaction of MPK38 and ASK1 appears to be increased by other ASK1 stimuli, including TNF-␣, endoplasmic reticulum stress (thapsigargin), and calcium overload (ionomycin) (Fig.  1E, upper right, and data not shown). We also determined the effect of ASK1 stimulators on the physical interaction between endogenous MPK38 and ASK1 in HEK293 cells. Exposure of the cells ASK1 stimuli, such as H 2 O 2 , TNF-␣, thapsigargin, and ionomycin resulted in a considerable increase in endogenous MPK38-ASK1 complex formation (Fig. 1E, lower, and data not shown), suggesting the involvement of MPK38 in the ASK1 signaling pathway.
Mapping of the Binding Domain Required for the MPK38-ASK1 Interaction-We performed in vivo binding assays to determine which domain of ASK1 contributes to MPK38 binding. The carboxyl-terminal regulatory domain (ASK1-C, amino acids 941-1375) of ASK1 was found to be responsible for MPK38 binding, whereas the amino-terminal (ASK1-N, amino acids 1-648) and kinase (ASK1-K, amino acids 649 -940) domains 293T cells transiently transfected with wild-type GST-MPK38 were incubated with increasing amounts of H 2 O 2 for 30 min, and MPK38 was purified on glutathione-Sepharose beads. The GST precipitates were subjected to an in vitro kinase assay using ZPR9 as a substrate, followed by SDS-PAGE and autoradiography (left). For H 2 O 2 time-dependent regulation of MPK38 and ASK1 kinase activity, 293T cells were treated with 2 mM H 2 O 2 for the indicated times. Cell lysates were immunoprecipitated by the indicated antibodies, and the kinase activities of MPK38 and ASK1 were determined by in vitro kinase assays using ZPR9 and MKK6(K82A) as substrates (right). The circled P-ZPR9 and P-MKK6(K82A) indicate the phosphorylated ZPR9 and MKK6(K82A), respectively. The relative level of kinase activity was quantitated by densitometric analyses, and fold increase relative to untreated samples was calculated. IP, immunoprecipitation; WB, Western blot. B, in vivo and in vitro association of MPK38 with ASK1. GST alone and GST-MPK38 were cotransfected with FLAG-ASK1 into 293T cells. GST fusion proteins were purified on glutathione-Sepharose beads (GST Purification), and the amounts of complex formation and FLAG-ASK1 used for the in vivo binding assay were determined by anti-FLAG antibody immunoblot (left). Cell lysates from HEK293 cells were subjected to immunoprecipitation using either rabbit preimmune serum (Preimm.) or anti-MPK38 antibody (␣-MPK38), followed by immunoblot analysis using an anti-ASK1 antibody to determine the complex formation between endogenous MPK38 and ASK1 (middle). As a control, the expression levels of MPK38 and ASK1 in the total cell lysate were analyzed by immunoblot using anti-MPK38 and anti-ASK1 antibodies, respectively. For native PAGE (8%) of the MPK38-ASK1 complex (right), purified recombinant MPK38 was autophosphorylated as described under "Materials and Methods." Autophosphorylated MPK38 (2-3 g) was incubated with unlabeled recombinant GST or GST-ASK1(K709R) (each 5 g) at room temperature for 1 h. C, effect of MPK38 and ASK1 kinase activities on MPK38-ASK1 association. HEK293 cells were transiently transfected with the appropriate expression plasmids, and MPK38-ASK1 complex formation was determined by Western blot analysis using anti-HA or anti-FLAG antibody as described in B. D, modulation of MPK38-ASK1(K709R), MPK38(K40R)-ASK1, or MPK38(K40R)-ASK1(K709R) complex formation by H 2 O 2 . HEK293 cells transfected with the indicated expression vectors were incubated with or without 2 mMH 2 O 2 for 30 min. The level of MPK38-ASK1 complex was analyzed by immunoblot using an anti-HA antibody. E, H 2 O 2 and TNF-␣modulation of MPK38-ASK1 interaction. HEK293 cells transfected with the expression vectors indicated were treated with 2 mM H 2 O 2 for 30 min or 500 ng/ml TNF-␣ for 30 min. Cell lysates were purified on glutathione-Sepharose beads (GST Purification) and immunoblotted with an anti-FLAG antibody (upper). The endogenous level of MPK38-ASK1 complex in the presence or absence of H 2 O 2 (or TNF-␣) was also analyzed by immunoblot using an anti-ASK1 antibody (lower). The relative level of MPK38-ASK1 complex formation was quantitated by densitometric analyses and fold increase relative to untreated samples expressing wild-type MPK38 and ASK1 was calculated (C-E).
were unable to bind with MPK38 ( Fig. 2A), indicating that ASK1 physically interacts with MPK38 through its carboxylterminal regulatory domain.
To identify the region of MPK38 required for ASK1 binding, we next performed in vivo binding assays with cells transfected with two deletion constructs MCAT, harboring the kinase catalytic domain (amino acids 7-269) of MPK38, and MPKC, comprising the carboxyl-terminal regulatory domain (amino acids 270 -643). Wild-type MPK38 and MPKC exhibited binding to ASK1, whereas the MCAT construct was not capable of ASK1 binding (Fig. 2B), indicating that the carboxyl-terminal regulatory domain of MPK38 was responsible for ASK1 binding. Together, these results demonstrate that the physical association between MPK38 and ASK1 is mediated through their carboxyl-terminal regulatory domains.
MPK38 Stimulates ASK1 Kinase Activity in a Kinase-dependent Manner-To establish the physiological role for the MPK38-ASK1 interaction, we investigated the effects of this interaction on ASK1 function. To determine whether MPK38 has an effect on ASK1 kinase activity, 293T cells were transiently transfected with ASK1 alone or cotransfected with MPK38. The recombinant MKK6(K82A) protein expressed in Escherichia coli was purified and used as a substrate for the ASK1 kinase assay. ASK1 kinase activity significantly increased when ASK1 was coexpressed with MPK38 ( Fig. 3A, left). In a separate experiment with recombinant wild-type ASK1, we also demonstrated that recombinant MPK38 proteins stim-ulatedASK1kinaseactivityinadosedependent manner (Fig. 3A, right). However, the stimulatory effect of recombinant MPK38 on ASK1 kinase activity was not observed in the presence of recombinant ASK1-K, which was unable to bind with MPK38 (Fig. 3A, middle).
To examine whether the activity of MPK38 was involved in the ASK1 activation, we analyzed the effect of the kinase-dead mutant of MPK38 (MPK38(K40R)) on ASK1 kinase activity using an in vitro kinase assay. Coexpression of kinase-dead MPK38 had no effect on the modulation of ASK1 kinase activity compared with expression of wild-type ASK1 alone (Fig. 3B, top panel, 1st lane versus 3rd lane). Similarly, the phosphorylation of Thr 838 of ASK1, which correlates with ASK1 activation (21), was not elevated by transfection with kinasedead MPK38 (Fig. 3B, 2nd panel). The levels of immunoprecipitated ASK1 proteins were analyzed, and similar expression levels were found for the ASK1 construct (Fig. 3B, 3rd panel), indicating that the observed differences in phosphorylated MKK6(K82A) were not because of differences in ASK1 expression levels of HA immunoprecipitates. Taken together, these experiments demonstrate that MPK38 may be a positive regulator of ASK1 activity.   Figs. 1 and 2), we next determined whether ASK1 can act as a substrate for MPK38. The recombinant nonphosphorylated form of the ASK1(K709R) or wild-type ASK1 protein was expressed in E. coli, purified, and used as substrates for the MPK38 kinase assay. Extracts from 293T cells expressing GST-MPK38 were purified with glutathione-Sepharose beads and incubated with [␥-32 P]ATP to allow phosphorylation of the recombinant ASK1(K709R) or wild-type ASK1. In addition to the increase in the phosphorylation of wild-type ASK1, ASK1(K709R) phosphorylation was observed in the presence of MPK38 (Fig. 4A, top panel), indicating that ASK1 may be a substrate for MPK38. A similar result was also observed in the phosphorylation of ASK1 Thr 838 (Fig. 4A, 2nd panel).
As the phosphorylation of Thr 838 of human ASK1 (Thr 845 in mice) correlates with ASK1 activation (21) and MPK38 stimulates ASK1 kinase activity (Fig. 3), we used an in vitro kinase assay using recombinant MPK38 to examine whether this and other (Ser 83 , Ser 967 , and Ser 1034 ) ASK1 phosphorylation sites play a role in MPK38-mediated phosphorylation. Mutation of ASK1(K709R) Thr 838 to Ala 838 completely abolished MPK38-dependent phosphorylation compared with the control with ASK1(K709R) as a substrate (Fig. 4B, top panel, 3rd lane versus 5th lane), indicating that the Thr 838 within the activation loop of ASK1 represents a potential phosphorylation site for MPK38. These results suggest that MPK38 directly phosphorylates ASK1 on Thr 838 through physical interaction and activates ASK1.

versus lane 5).
These results suggest that, despite physical binding, MPK38 may not be a substrate for ASK1. Based on this, together with the above results obtained in Fig. 4, A and B, we reasoned that MPK38 may be an upstream kinase that activates ASK1. To examine this hypothesis, MPK38 was purified on glutathione-Sepharose beads using HEK293 cell extracts expressing GST-MPK38. MPK38 kinase activity was determined by in vitro kinase assays using recombinant ASK1(K709R), MKK6(K82A), or p38 as substrates. MPK38 phosphorylated recombinant ASK1(K709R), whereas phosphorylation of recombinant MKK6(K82A) and p38 by MPK38 was not detectable (Fig. 4C). These findings suggest that MPK38 may act as an upstream regulator of the MAPKKK ASK1.
MPK38 Increases the Interaction between ASK1 and Its Substrate MKK3-We next examined whether MPK38 contributes to the interaction between ASK1 and its substrate MKK3. HEK293 cells transfected with vectors expressing FLAG-ASK1 and HA-MKK3 in the presence or absence of MPK38 were subjected to immunoprecipitation using an anti-HA antibody, followed by immunoblot analysis using an anti-FLAG antibody. There was a significant increase in complex formation between ASK1 and MKK3 in cells coexpressing MPK38 compared with expression in the absence of MPK38 (Fig. 5A). In contrast, no difference in complex formation was observed in the presence of N-acetyl-L-cysteine (Nac), a potent antioxidant (Fig. 5B). This indicates that intracellular redox regulation might be involved in MPK38mediated increase of the association between ASK1 and MKK3. We also observed a similar trend showing the importance of the phosphorylation of Thr 838 of ASK1 in MPK38mediated modulation of the complex formation between ASK1 and MKK3 (Fig. 5C). Consistent with this, knockdown of endogenous MPK38 decreased the complex formation between ASK1 and MKK3, although this reduction was overcome by expressing a wobble mutant of MPK38 (Fig. 5D).  (2nd panel). The expression levels of immunoprecipitated ASK1 and wild-type and kinase-dead MPK38 in total cell lysates were analyzed using anti-HA and anti-GST antibodies, respectively (3rd and 4th panels). The relative level of ASK1 kinase activity was quantitated by densitometric analyses, and fold increase relative to control expressing ASK1 or ASK1-K alone was calculated. re., recombinant.
These results suggested that ASK1 activation by MPK38 increased the interaction between ASK1 and its substrate.
MPK38 Stimulates Signaling Downstream of ASK1-We examined whether MPK38 affects signaling downstream of ASK1, as Hsp72, another interacting partner of ASK1, was previously shown to modulate signaling downstream of ASK1 through its direct interaction with ASK1 (6). Parental SK-N-BE(2)C cells or SK-N-BE(2)C cells stably expressing pCMV-MPK38 (SK-MPK38) were treated with H 2 O 2 and subsequently immunoprecipitated using anti-ASK1, anti-MKK3, and anti-p38 antibodies. Endogenous ASK1, MKK3, and p38 kinase activities were evaluated using in vitro kinase assays. H 2 O 2 sufficiently stimulated the kinase activities of ASK1, MKK3, and p38 in parental SK-N-BE(2)C cells, and these effects were further enhanced by overexpression of MPK38 (Fig.  6A, SK-MPK38, upper panels). A similar result was also observed in immunoblot analysis using antiphospho-specific antibodies for ASK1 Thr 838 , MKK3/6, p38, and ATF2 (Fig. 6A, lower left). Consistent with this, knockdown of endogenous MPK38 showed an opposite trend in the modulation of ASK1, MKK3, and p38 kinase activities (Fig. 6B), suggesting that MPK38 stimulates ASK1-mediated signaling to p38 kinase.
To determine whether MPK38 targets ASK1 directly, we also performed in vitro kinase assays of ASK1, MKK3, MKK6, and p38 in the presence or absence of recombinant MPK38. HEK293 cells were transiently transfected with HA-ASK1, HA-MKK3, HA-MKK6, or HA-p38 and treated with or without H 2 O 2 . The cells were then subjected to immunoprecipitation with an anti-HA antibody. The resulting immunoprecipitates were assayed for kinase activities of ASK1, MKK3, MKK6, or p38 in the presence or absence of recombinant MPK38. MPK38 specifically stimulated ASK kinase activity but had no effect on the kinase activities of MKK3, MKK6, or p38 (supplemental Fig. S4). These results suggest that MPK38 enhances ASK1-mediated signaling through direct stimulation of ASK1.
MPK38 Stimulates ASK1-mediated AP-1 Transcriptional Activity in a Kinase-dependent Manner-ASK1 is a MAPKKK involved in the activation of JNK/stress-activated protein kinase and p38 MAPK (2,24). Because AP-1 is a transcription factor activated by JNK and p38 kinases, we used an AP-1 luciferase reporter to determine whether MPK38 affects ASK1-mediated transactivation. Wild-type MPK38 (MPK38(WT)) significantly increased ASK1-mediated AP-1 transcriptional activity in a dose-dependent manner, whereas kinase-dead MPK38 had no effect (Fig. 7A). We also confirmed the roles of MPK38 in ASK1-mediated transactivation using the MPK38  (2nd panel). The position and expression levels of GST-MPK38, re.ASK1(K709R), and re.ASK1(WT) were monitored by immunoblotting with an anti-GST antibody (3rd and bottom panels). The relative level of kinase activity was quantitated by densitometric analyses, and fold increase relative to control containing recombinant wild-type ASK1 alone was calculated. WB, Western blot. B, identification of MPK38 phosphorylation sites on ASK1. An in vitro kinase assay was performed with 5 g of recombinant ASK1(K709R) or one of its substitution mutants (ASK1(S83A), ASK1(T838A), ASK1(S967A), or ASK1(S1034A)), as well as recombinant wild-type MPK38 (10 g), as described in A (top panel). The phosphorylation of ASK1 Thr 838 and expression of recombinant MPK38 and ASK1 were determined by immunoblotting with anti-phospho-ASK1(T845) and anti-GST antibodies, respectively (2nd to bottom panels). The relative level of kinase activity was quantitated by densitometric analyses, and fold increase relative to control samples containing ASK1(K709R) was calculated. C, after 48 h of transfection with GST-MPK38, HEK293 cell lysates were subjected to precipitation with glutathione-Sepharose beads (GST Purification) and then analyzed by in vitro kinase assays with recombinant ASK1(K709R), MKK6(K82A), and p38 as substrates. The circled P-ASK1(K709R) and circled P-MPK38 indicate the phosphorylated ASK1(K709R) and autophosphorylated MPK38, respectively. re., recombinant.
knockdown system. Transfection of MPK38 siRNA resulted in a significant decrease in ASK1-mediated AP-1 transcriptional activity that was proportional to the amount of MPK38 siRNA transfected (Fig. 7A, 4th lane versus 7th and 8th lanes).
Next, to test the effect of MPK38 on ASK1-mediated JNK activation, HEK293 cells were transfected with vectors expressing GST-JNK and HA-ASK1 in the presence or absence of wildtype and kinase-dead MPK38. JNK activity was evaluated using an in vitro kinase assay with c-Jun as a substrate. As expected, ASK1-mediated JNK activation was markedly increased by wild-type MPK38 in a dose-dependent manner, whereas kinase-dead MPK38 had no effect on ASK1-mediated JNK activation (Fig. 7B, left). To verify whether the knockdown of endogenous MPK38 contributed to altered ASK1-mediated JNK activation, HEK293 cells transfected with GST-JNK and HA-ASK1, together with MPK38-specific siRNA, were purified on glutathione-Sepharose beads, followed by an in vitro kinase assay using c-Jun as a substrate. ASK1-mediated JNK activation was decreased in a dose-dependent manner in MPK38-knockdown cells compared with control cells expressing JNK and ASK1 in the absence of MPK38 siRNA (Fig. 7B, middle, 4th lane  versus 7th and 8th lanes). A similar result was also observed in HaCaT cells stably expressing an shRNA targeting MPK38 (MPK38(KD)) (Fig. 7B, right). These results suggest that ASK1 phosphorylation that is mediated via direct interaction with MPK38 increases ASK1-mediated signaling to both JNK and p38 kinases.
MPK38 Stimulates H 2 O 2 -mediated Apoptosis-Because MPK38 interacts with ASK1 (see Figs. 1 and 2) and the ASK1 activation induces apoptotic cell death under various conditions (2, 3), we next examined whether MPK38 could influence H 2 O 2 -mediated cell death. Expression of wild-type MPK38 in HEK293 cells resulted in a significant increase in H 2 O 2 -induced cell death in a dose-dependent manner, as determined by GFP system and TUNEL staining (Fig. 8). This indicated that MPK38 is involved in H 2 O 2 -mediated cell death. We also deter- We also performed knockdown experiments of MPK38 using MPK38 siRNA. Transfection of HEK293 cells with siRNA duplexes targeting MPK38 resulted in a significant decrease of H 2 O 2 -mediated apoptosis, proportional to the amount of were treated with 2 mM H 2 O 2 for 30 min. The immunoprecipitates were also subjected to in vitro kinase assays as described in A. ASK1 Thr 838 , MKK3/6, p38, and ATF2 phosphorylation were determined by immunoblot analysis using the indicated phospho-specific antibodies (A and B, lower, left panels). The expression levels of ASK1, MKK3, p38, ATF2, and MPK38 in total cell lysates were analyzed by immunoblot using the indicated antibodies (A and B, lower, right panels). The relative level of kinase activity was quantitated by densitometric analyses, and fold increase relative to untreated samples in parental cells was calculated. B, enhancement of JNK activity by MPK38. HEK293 cells were cotransfected using GST-JNK (1.5 g) and HA-ASK1 (2 g) in the presence or absence of increasing amounts of wild-type (WT) and kinase-dead (K40R) forms of MPK38 (6 and 9 g) or MPK38 siRNA 1 (100 and 200 nM). An in vitro kinase assay for JNK activity was performed as described under "Materials and Methods." The amounts of precipitated JNK and the expression level of ASK1 and MPK38 in total cell lysates were determined by immunoblot analysis using anti-GST and anti-HA antibodies. MPK38(KD) cells transfected with GST-JNK and HA-ASK1 were also precipitated using glutathione-Sepharose beads (GST Purification), and the precipitates were subjected to an in vitro kinase assay using c-Jun as a substrate to determine JNK activity (right). The relative level of JNK activity was quantitated by densitometric analyses, and fold increase relative to control HEK293 cells expressing JNK alone or untreated HaCaT cells expressing JNK and ASK1 was calculated. WB, Western blot. siRNA used in the transfection (Fig. 8, 6th lane versus 13th and  14th lanes). These data strongly suggest that MPK38 contributes to H 2 O 2 -mediated apoptosis by enhancing the ASK1 activity through phosphorylation.
ASK1 has been implicated in caspase-3 activation (25,26). PARP is known to be as an in vivo substrate of capase-3 (27). Based on this, to investigate whether the stimulation of H 2 O 2mediated apoptosis by MPK38 is dependent on its activation of caspase-3, leading to the proteolytic cleavage of PARP, we assessed the effects of MPK38 on H 2 O 2 -induced caspase-3 activity and PARP cleavage by immunoblotting with anticaspase-3 and anti-PARP antibodies, respectively. Results indicated that MPK38 stimulated H 2 O 2 -mediated apoptosis by upregulating an in vivo capase-3 activity (supplemental Fig. S6).

DISCUSSION
The mechanism for ASK1 activation in response to apoptotic stimuli has not been fully elucidated, despite the proposed function of ASK1 in mediating multiple cell death pathways. Recent studies have demonstrated that ASK1 activity is positively or negatively regulated by its interacting partners, including tumor necrosis factor receptor-associated factor (28), Daxx (1), JNK/stress-activated protein kinase-associated protein 1 (JSAP1)/JNK-interacting protein 3 (JIP3) (29), thioredoxin (3,4), glutaredoxin (7), HSP72 (6), Raf-1 (3), Akt/PKB (30), PP5 (5), and 14-3-3 (8). In addition, ASK1 phosphorylation also regulates its activity (5,21,30,31). Akt/PKB binds to and phosphorylates Ser 83 of ASK1, resulting in the inhibition of ASK1-mediated apoptosis (30); and 14-3-3 interacts with phosphorylated Ser 967 of ASK1 to inhibit ASK1 function (8). Conversely, PP5 dephosphorylates murine Thr 845 within the activation loop of ASK1 that is critical for ASK1 activation and thereby inhibits ASK1-mediated apoptosis (5). Although Akt/PKB was recently described as a kinase that is critical for ASK1 Ser 83 phosphorylation (30), the question remains which kinases are responsible for ASK1 phosphorylation at multiple sites, including Thr 845 , Ser 967 , and Ser 1034 . To gain insight into the mechanism(s) by which MPK38 positively regulates ASK1 activity, we examined whether the Thr 838 (corresponding to Thr 845 in mice) plays a key role in MPK38-mediated phosphorylation using an in vitro kinase assay and immunoblot analysis. MPK38 was shown to phosphorylate Thr 838 of ASK1, but not Ser 83 , Ser 967 , and Ser 1034 (Fig. 4B). In addition, the previous finding of Thr 845 phosphorylation in kinase-dead ASK1 of H 2 O 2 -treated ASK1-null mouse embryonic fibroblasts strongly supports the notion that Thr 845 phosphorylation occurs via a putative Thr 845 kinase in cells (21). A recent report (32) showing that the ASK1interacting partner ASK2 activates ASK1 through Thr 838 phosphorylation is interesting in regards to the search for this putative kinase. We examined whether MPK38 functions as an upstream kinase of ASK1 or another MAPKKK, because recent studies showed that ASK1 associated with other MAPKKKs, including TAK1 (33) and Raf-1 (34). These interactions lead to ASK1mediated inhibition of TAK1-mediated NF-B signaling as well as Raf-1-mediated inhibition of ASK1 kinase activity. As shown in Fig. 4C, MPK38 was able to phosphorylate GST-ASK1(K709R), but not GST-MKK6(K82A) or GST-p38. Furthermore, neither ASK1 nor TAK1 could phosphorylate GST-MPK38(K40R) in a reciprocal way (see supplemental Fig. S1 and data not shown). These results strongly suggest that MPK38, unlike ASK2 (32), is apparently an upstream kinase of ASK1 but not of other MAPKKKs. In addition, MPK38 and ASK2 are not like each other in terms of their overall structures; MPK38 possesses an amino-terminal kinase domain and a carboxyl-terminal regulatory domain (9), but ASK2 contains a kinase domain in the middle portion with amino-and carboxylterminal flanking regions (32). Furthermore, there is only 29% amino acid identity between MPK38 and ASK2, particularly in the kinase domain. Based on this, we speculate that MPK38 and ASK2 work in different ways to regulate ASK1 activity, despite the ability of these two kinases to phosphorylate the same site (Thr 838 /Thr 845 ) of ASK1. However, we cannot rule out the possibility that ASK1 activation can be also triggered by ASK1 oligomerization, which is independent of direct phosphorylation of Thr 845 through putative Thr 845 kinases.
Little is known about the biological functions or the molecular mechanism(s) underlying MPK38 activity, despite its implicated involvement in various cellular processes, including cell cycle, spliceosome assembly, carcinogenesis, and self-renewal of stem cells (35). The results presented here suggest that MPK38 stimulates the activation of ASK1-mediated signaling to both JNK and p38 kinases through direct interaction with and phosphorylation of ASK1, and that the MPK38-ASK1 interaction may provide a molecular basis for the proposed several MPK38 functions. Moreover, the role of MPK38 as an inducer of ASK1-mediated signaling may clarify the mechanism(s) underlying MPK38-mediated signaling in carcinogen- FIGURE 8. Effect of MPK38 on H 2 O 2 -mediated apoptosis. HEK293 cells were transiently transfected with increasing amounts of wild-type MPK38 (0.8 and 1.6 g) and ASK1 (0.5 and 1 g) or MPK38 and ASK1 siRNAs (100 and 200 nM) in the presence or absence of H 2 O 2 . Apoptotic cell death was determined using the GFP expression system (GFP) or TUNEL. Cells exposed to 1 mM H 2 O 2 for 9 h was used as a positive control. Data shown are means (ϮS.E.) of three independent experiments. p Ͻ 0.05 relative to control; significance calculated by Student's t test.
esis. We have revealed that MPK38 directly binds to and activates ASK1 to stimulate ASK1-mediated signaling through ASK1 phosphorylation at Thr 838 , and our results suggest that MPK38 is a potential upstream kinase of ASK1. In addition, H 2 O 2 -dependent modulation of MPK38 activity may provide an effective way to study the biological role of MPK38 in detail.