A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3.

A cDNA encoding a novel member of the mitogen-activated protein kinase kinase (MAPKK) family, MAPKK6, was isolated and found to encode a protein of 334 amino acids, with a calculated molecular mass of 37 kDa that is 79% identical to MKK3. MAPKK6 was shown to phosphorylate and specifically activate the p38/MPK2 subgroup of the mitogen-activated protein kinase superfamily and could be demonstrated to be phosphorylated and activated in vitro by TAK1, a recently identified MAPKK kinase. MKK3 was also shown to be a good substrate for TAK1 in vitro. Furthermore, when co-expressed with TAK1 in cells in culture, both MAPKK6 and MKK3 were strongly activated. In addition, co-expression of TAK1 and p38/MPK2 in cells resulted in activation of p38/MPK2. These results indicate the existence of a novel kinase cascade consisting of TAK1, MAPKK6/MKK3, and p38/MPK2.

Here we report cDNA cloning and characterization of a novel member of MAPKK, tentatively called MAPKK6, with 79% identity to MKK3, which specifically activates p38/MPK2. Our studies in vitro and in various cells further suggest that TAK1 (20), a recently identified, novel member of the MAPKKK family, can work as a direct activator for both MAPKK6 and MKK3. In addition, co-expression of TAK1 and p38/MPK2 resulted in activation of p38/MPK2, suggesting the existence of a novel kinase cascade consisting of TAK1, MAPKK6/MKK3, and p38/MPK2.

MATERIALS AND METHODS
cDNA Cloning and Sequence Analysis-Patient EY has a long history of Behcet disease and has an antiserum that reacts with nuclear and cytosolic proteins of HEp-2 cells. EY serum was used for immunoscreening of 5 ϫ 10 5 recombinants from a HeLa Zap cDNA library (Clontech). Three independent clones were isolated and subcloned in vivo into the pBluescript plasmid using the R408 helper phage. Their nucleotide sequences were determined by dye terminator sequencing with an Applied Biosystems model 373A machine.
Hybridization Analysis-Northern blots were performed using 2 g of poly(A) ϩ RNAs isolated from different human tissues, fractionated by denaturing agarose gel electrophoresis, and transferred onto nylon membranes (Clontech). The blots were hybridized to a synthesized oligonucleotide probe (50 base pairs) designed from the sequence of the clone 4-3 and labeled with terminal deoxynucleotidyltransferase (Toyobo) and [␣-32 P]CTP (Amersham Corp.). The integrity of the mRNA was confirmed by hybridization to an actin probe.
Preparation of Recombinant Proteins-Human p38 (21) and human MKK3 (11) coding regions were amplified by the polymerase chain reaction, using human skeletal muscle cDNA (Clontech) as a template and the following oligonucleotide probes: 5Ј-GGCCGGATCCATGTCT-CAGGAGAGGCCCAC-3Ј and 5ЈGCGCGGATCCTCAGGACTCCATC-TCTTCTT-3Ј for p38, and 5Ј-GGCCAGATCTATGTCCAAGCCACCCG-CACC-3Ј and 5Ј-GCGCAGATCTCTATGAGTCTTCTCCCAGGA-3Ј for MKK3. MAPKK6 cDNA was subcloned into the SalI/HindIII sites of pQE31 (Qiagen Inc.). p38 cDNA was subcloned into the BamHI site of pET28a (Novagen). MKK3 cDNA was subcloned into the BamHI site of pGEX-2T (Pharmacia Biotech Inc.). The genes were expressed in a bacteria strain BL21(DE3)pLysS as His-tagged or GST-fusion proteins and purified according to manufacturer's instructions. A kinase-negative (KNϪ) MPK2(K54R) and wild type SAPK␣ were expressed as His-tagged proteins and purified using a Ni 2ϩ -affinity column (18). Wild type Xenopus MAPK (22) was subcloned into pTrcHisC (Invitrogen) and expressed in the bacteria strain BL21(DE3)pLysS as a His-tagged protein. Recombinant c-Jun was expressed and purified as * This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan, the Naito Memorial Science Foundation, the Toray Science Foundation, and the Mitsubishi Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) D84440.
§ These authors contributed equally to this work. noprecipitation with anti-HA monoclonal antibody (12CA5) as described previously (20). The immunoprecipitated Raf-1 was capable of activating Xenopus MAPKK (data not shown). The immunoprecipitates were incubated with MAPKK6 or MKK3 (20 g/ml) and 100 M [␥-32 P]ATP (1 Ci) in the absence (for detecting the ability to phosphorylate MAPKKs) or presence (for detecting the ability to activate MAPKKs) of KNϪMPK2 (100 g/ml) in a solution containing 20 mM Tris-Cl, pH 7.5, 2 mM EGTA, and 10 mM MgCl 2 for 30 min at 30°C. To detect the ability to activate MAPKK6, the immunoprecipitate was first incubated with MAPKK6 (20 g/ml) in a solution containing 100 M ATP, 20 mM Tris-Cl, pH 7.5, 2 mM EGTA, and 10 mM MgCl 2 for 20 min at 30°C, then KNϪMPK2 (final concentration, 100 g/ml) and 1 Ci of [␥-32 P]ATP were added, and the reaction mixture was incubated for a further 10 min at 30°C. The reaction was stopped by the addition of Laemmli's sample buffer. The radioactivity incorporated into recombinant MAPKK6, MKK3, and KNϪMPK2 was detected by autoradiography.
Cell Cultures and Transfection-COS7 cells were cultured at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Mv1Lu cells were cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 5% fetal bovine serum. When needed, cells were transfected using LipofectAMINE according to the manufacturer's instructions (Life Technologies Inc.). For preparing lysates, cells were washed once with ice-cold Hepes-buffered saline and lysed in a lysis buffer containing 20 mM Hepes, pH 7.35, 12.5 mM 2-glycerophosphate, 150 mM NaCl, 1.5 mM MgCl 2 , 2 mM EGTA, 10 mM NaF, 0.5% Triton X-100, 2 mM dithiothreitol, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and 20 g/ml aprotinin. Lysates were clarified by centrifugation at 15,000 ϫ g for 15 min. For immunoprecipitation, the supernatants were incubated with 12CA5 for 1 h at 4°C with rocking. After the addition of protein A-Sepharose (Pharmacia Biotech Inc.) beads, the lysates were incubated for an additional 1 h. The beads were washed three times with ice-cold phosphate-buffered saline and subjected to kinase assays.
Protein Kinase Assays-To assay the ability to activate MAPK, SAPK, or p38, samples were incubated for 30 min at 30°C with 0.5 g of His-tagged MAPK, SAPK, or p38 in a final volume of 10 l of solution containing 100 M ATP, 20 mM Tris-Cl, pH 7.5, 2 mM EGTA, and 15 mM MgCl 2 . Then labeled ATP ([␥-32 P]ATP, 1 Ci) and 3 g of MBP, c-Jun, or ATF2 were added to the reaction mixture, and incubation was carried out for the indicated times at 30°C in a final volume of 15 l. To assay the ability to phosphorylate KNϪMPK2, samples were incubated for 30 min at 30°C with 3 g of KNϪMPK2 in a solution containing 100 M [␥-32 P]ATP (3 Ci), 20 mM Tris-Cl, pH 7.5, 2 mM EGTA, and 15 mM MgCl 2 . The reaction was stopped by addition of Laemmli's sample buffer and boiling. After SDS-polyacrylamide gel electrophoresis, phosphorylation of these proteins was detected by autoradiography and quantified using an image analyzer (Fujix BAS2000).
Immunofluorescence-Transfected Mv1Lu cells were washed with PBS, fixed by incubation for 5 min in 3.7% formaldehyde in PBS, and permeabilized in PBS containing 0.5% Triton X-100. After incubation for 1 h at room temperature with anti-HA antibody at a 1:500 dilution in PBS, the cells were washed three times with PBS and incubated for 1 h at room temperature with fluorescein-conjugated secondary antibody (Zymed Laboratories, Inc.) at a 1:50 dilution in PBS. Then, cells were washed three times with PBS.

RESULTS AND DISCUSSION
One of 3 clones isolated by screening a human HeLa cell cDNA library with patient EY autoantiserum encoded a novel protein kinase. The cDNA contains an open reading frame encoding a protein of 334 amino acids with a calculated molecular mass of 37 kDa (Fig. 1A). The deduced amino acid sequence contains all the amino acids that are conserved among protein kinases (24) and shows striking similarity to those of the MAPKK superfamily, 79% identity to human MKK3 (11), Then the activities of MAPK, SAPK, and p38 were measured by using MBP, c-Jun, and ATF2 as substrates, respectively. After SDS-polyacrylamide gel electrophoresis, the radioactivity of the MBP, c-Jun, and ATF2 bands was detected by autoradiography (lower panel and data not shown) and quantified using an image analyzer (Fujix BAS2000) (results shown in upper panel). The radioactivity incorporated into p38 was also detected by autoradiography and shown here. 37% identity to human MKK1 (25), 34% identity to human MKK2 (26), 52% identity to human MKK4 (11), and 38% identity to human MEK5 (15) (Fig. 1A).
Thus, this novel kinase appears to belong to the MAPKK superfamily and has been tentatively named MAPKK6. Northern blot analysis showed that MAPKK6 is expressed most highly in skeletal muscle and moderately in thymus, small intestine, and pancreas (Fig. 1B). When an HA-tagged MAPKK6 was expressed in cells, the distribution was throughout the cytoplasm (Fig. 1C). Thus nuclear staining of HEp-2 cells may be attributable to the other clones (nucleoporin (NUP358) and an unidentified human cDNA clone 42678) simultaneously isolated by this immunoscreening with EY serum (data not shown).
To examine whether MAPKK6 is able to activate members of the MAPK superfamily, MAPKK6 was expressed as a Histagged protein in bacteria and purified. Subgroups of the MAPK superfamily (Xenopus classical MAPK, rat SAPK␣, and human p38) were also expressed in bacteria and purified. When the purified MAPKK6 was incubated with MAPK, SAPK, or p38 in the presence of ATP, only p38 was heavily phosphorylated (Fig. 2, lower panel); the level of phosphorylation of MAPK or SAPK was below 1% of that of p38 (data not shown). Thus, only the activity of p38 was specifically and strongly stimulated by incubation with MAPKK6 (Fig. 2, upper panel, and data not shown). In these experiments, MBP, c-Jun, and ATF2 were used as substrates for MAPK, SAPK, and p38, respectively (Fig. 2). The results clearly indicated that MAPKK6 is a specific activator for the p38/MPK2 subgroup of the MAPK superfamily. As MKK3 was earlier shown to be a specific activator of p38 (11), MAPKK6 may be most closely related to MKK3 in terms of both structure and substrate specificity.
We have recently identified TAK1, a novel member of the MAPKKK family, that may function in the transforming growth factor-␤ (TGF␤) signaling pathway (20). Since TAK1 does not activate classical MAPKK, we considered the possibility that MKK3 and MAPKK6 could be its natural substrates. To address this possibility, activated TAK1 was expressed in yeast, purified by immunoprecipitation, and incubated with bacterially expressed MAPKK6 in the presence of [␥-32 P]ATP. MAPKK6 was found to be heavily phosphorylated (Fig. 3A, lane  2). When the incubation was performed in the absence of TAK1 (Fig. 3A, lane 1) or with a kinase-inactive mutant of TAK1 instead of the activated TAK1 (Fig. 3A, lane 3), no phosphorylation of MAPKK6 was observed, indicating that the catalysis was by TAK1 itself. When Raf-1 (Fig. 3A, lane 4) or MEKK1 (data not shown) was used as the kinase, no phosphorylation of MAPKK6 was detected, although both Raf-1 and MEKK1 could efficiently phosphorylate classical MAPKK and/or MKK4. Therefore, MAPKK6 was phosphorylated by TAK1 but not by Raf-1 or MEKK1, the resultant form demonstrating a much higher kinase activity toward the kinase-inactive form of p38/ MPK2 than unphosphorylated MAPKK6 (Fig. 3B). Thus, MAPKK6 was established to be functionally activated by TAK1-mediated phosphorylation. The same kind of experiments with MKK3 revealed similar TAK1-mediated phosphorylation (Fig. 3C) and activation (Fig. 3D). We conclude, from these results, that TAK1 is a direct activator of both MAPKK6 and MKK3 in vitro. As far as we know, this is the first identification of an upstream kinase for MKK3.
To examine whether TAK1 can work as an activator in cells, MAPKK6 or MKK3 was expressed together with active TAK1 or kinase-inactive TAK1 in COS7 cells. The expressed MAPKK6 or MKK3 was greatly activated by co-expression of active TAK1 (Fig. 4A) but not by co-expression of inactive TAK1 (Fig. 4A and data not shown). These results taken together suggest that TAK1 can work as a direct activator of MAPKK6 and MKK3, two specific activators of the p38/MPK2 subgroup of the MAPK superfamily. Consistent with this suggestion, co-expression of active TAK1 and p38 in COS7 cells resulted in activation of p38 (Fig. 4A). Thus, this study established the existence of a kinase cascade consisting of TAK1, MAPKK6/ MKK3, and p38. Since TAK1 was earlier shown to be activated by treatment with TGF-␤ (20), we also examined whether MAPKK6 or MKK3 was affected by this growth factor. Both MAPKK6 (Fig. 4B) and MKK3 (data not shown) were indeed activated, but the extent was not as large as that activated with exposure to hyperosmolarity (Fig. 4B). This might be interpreted as suggesting the existence of an as yet unidentified member of the MAPKK superfamily, which is a better substrate for TAK1 than MAPKK6 or MKK3. Conversely, it is possible that there are as yet unidentified members of the MAPKKK family lying upstream of MAPKK6 and MKK3.
In summary, this study identified a novel kinase cascade consisting of TAK1, MAPKK6/MKK3, and p38, by identifying a novel MAPKK member, MAPKK6, and revealing activation of both MAPKK6 and MKK3 by TAK1, a newly identified MAP-KKK. Further work is needed to elucidate the functions and regulatory mechanisms of this kinase cascade in vivo.
Addendum-While this manuscript was under review, Raingeaud et al. (27) and Han et al. (28) reported cDNA cloning of MKK6 and MKK6b, respectively, which are identical to MAPKK6 in this paper. After 24 h, the cells were collected, HA-MAPKK6, HA-MKK3, or HA-p38 was immunoprecipitated, and the activity was measured as described under "Materials and Methods." Substrate phosphorylation was demonstrated by autoradiography. B, Mv1Lu cells transiently transfected with pSR␣-HA-MAPKK6 were stimulated with 20 ng/ml TGF-␤1 for 15 min or 0.7 M NaCl for 30 min. HA-MAPKK6 was immunoprecipitated, and its activity was determined. The representative autoradiogram illustrated is the result of two separate experiments and the -fold increase of kinase activity is indicated below each lane.