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Selective Activation of p38 Mitogen-activated Protein (MAP) Kinase Isoforms by the MAP Kinase Kinases MKK3 and MKK6

Selective Activation of p38 Mitogen-activated Protein (MAP) Kinase Isoforms by the MAP Kinase... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 3, Issue of January 16, pp. 1741–1748, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Selective Activation of p38 Mitogen-activated Protein (MAP) Kinase Isoforms by the MAP Kinase Kinases MKK3 and MKK6* (Received for publication, July 28, 1997, and in revised form, October 27, 1997) Herve ´ Enslen, Joe ¨ l Raingeaud, and Roger J. Davis‡ From the Howard Hughes Medical Institute, Program in Molecular Medicine, and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 The cellular response to treatment with proinflamma- and IL-7 (7), and the stress-induced transcription of c-jun and tory cytokines or exposure to environmental stress is c-fos in fibroblasts (8). The targets of p38 MAP kinase that mediated, in part, by the p38 group of mitogen-activated mediate these responses are poorly characterized. However, protein (MAP) kinases. We report the molecular cloning biochemical studies indicate that p38 MAP kinase signaling b2. This p38 of a novel isoform of p38 MAP kinase, p38 pathway activates the transcription factors CREB and ATF1 a, is inhibited by the pyridinyl im- MAP kinase, like p38 (9, 10), ATF2 (11, 12), CHOP (13), and MEF-2C (14). The p38 idazole drug SB203580. The p38 MAP kinase kinase MAP kinase also activates other protein kinases, such as Map- a, MKK6 is identified as a common activator of p38 kap-2 (15–17), Mapkap-3 (18, 19), and Mnk1/2 (20, 21). b2, and p38g MAP kinase isoforms, while MKK3 ac- p38 The p38 MAP kinase group includes the isoforms p38a (4, 12, a and p38g MAP kinase isoforms. The tivates only p38 16, 17, 22), p38b (23), and p38g (24 –27). Recent studies indi- MKK3 and MKK6 signal transduction pathways are cate the presence of a fourth p38 MAP kinase isoform, p38d (28, therefore coupled to distinct, but overlapping, groups of 29). These p38 MAP kinases are widely expressed in many p38 MAP kinases. tissues and are activated by dual phosphorylation on Thr and Tyr within the motif Thr-Gly-Tyr located in kinase subdomain VIII (12). This phosphorylation is mediated by a protein kinase Mitogen-activated protein (MAP) kinases are proline-di- cascade (1). Components of this signaling pathway include the rected protein kinases that mediate the effects of numerous MAP kinase kinases MKK3 (30) and MKK6 (11, 31–33). It is extracellular stimuli on a wide array of biological processes, also possible that MKK4 contributes to the activation of p38 such as cellular proliferation, differentiation, and death (1). MAP kinase. In vitro studies demonstrate that MKK4 activates Three groups of mammalian MAP kinases have been studied in both JNK and p38 MAP kinases (30, 34). However, the role of detail: the extracellular signal-regulated kinases (ERK) (2), the MKK4 as an activator of p38 MAP kinase in vivo is unclear c-Jun NH -terminal kinases (JNK) (3), and the p38 MAP ki- (35). nases (3). The ERKs are robustly activated by growth factors The activation of MKK3 and MKK6 is regulated by phospho- and phorbol ester, but are only weakly activated by cytokines rylation on Ser and Thr residues within subdomain VIII by and environmental stress. In contrast, JNK and p38 MAP MAP kinase kinase kinases (MKKK) (1). Further studies are kinases are strongly activated by cytokines and environmental required to define the function and specificity of MKKKs that stress, but are poorly activated by growth factors and phorbol ester. cause activation of the p38 MAP kinase pathway. However, one candidate MKKK for the p38 MAP kinase signaling pathway is Recently, progress toward understanding the physiological role of the p38 MAP kinases has been achieved through the use TAK1, which has been reported to activate MKK3 and MKK6 of drugs that bind p38 MAP kinase (4, 5). These drugs are (36 –38). Other MKKKs, which activate both JNK and p38 pyridinyl imidazole derivatives that inhibit p38 MAP kinase MAP kinases, include ASK-1 (39) and the mixed-lineage kinase activity (4, 5). Studies using these drugs indicate that p38 MAP MLK-3 (40 – 42). Other MKKKs that activate the JNK signal- kinase is required for lipopolysaccharide-induced production of ing pathway, for example MEKK1, do not cause activation of IL-1 and TNF in monocytes (4), the induction of IL-6 and p38 MAP kinase (30, 34). granulocyte-macrophage/colony-stimulating factor transcrip- The expression of multiple p38 MAP kinase isoforms in tion by TNF (6), the proliferation of T cells in response to IL-2 mammalian tissues suggests that these MAP kinases may dif- fer in their physiological function. These p38 MAP kinases may be coupled to different upstream signaling pathways. This * These studies were supported in part by National Institutes of would enable the activation of specific p38 MAP kinase iso- Health, NCI, Grants CA65861 and CA72009. The costs of publication of forms in response to different stimuli. Alternatively, these p38 this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance MAP kinase isoforms may differ in their substrate specificity. with 18 U.S.C. Section 1734 solely to indicate this fact. Such differences could allow coupling of different p38 MAP The nucleotide sequence(s) reported in this paper has been submitted TM kinase isoforms to different signal transduction targets. to the GenBank /EBI Data Bank with accession number(s) AF031135. ‡ Investigator of the Howard Hughes Medical Institute. To whom The purpose of this study was to examine the p38b MAP correspondence should be addressed: Howard Hughes Medical Insti- kinase signal transduction pathway. We find that p38b MAP tute, Program in Molecular Medicine, University of Massachusetts kinase (23) is not a functional protein kinase in vitro or in vivo. Medical School, 373 Plantation St., Worcester, MA 01605. However, a novel p38b MAP kinase isoform (p38b2) that was The abbreviations used are: MAP, mitogen-activated protein; MKK, MAP kinase kinase; MKKK, MAP kinase kinase kinase; ERK, extra- isolated from a human brain cDNA library encoded a functional cellular signal-regulated kinase; JNK, c-Jun NH -terminal kinase; IL, 2 protein kinase. This novel p38 MAP kinase isoform was inhib- interleukin; TNF, tumor necrosis factor; GST, glutathione S-transfer- ited by pyridinyl imidazole drugs. The MAP kinase kinase ase; RT, reverse transcriptase; PCR, polymerase chain reaction; PAGE, MKK6 activated p38a, p38b2, and p38g, while MKK3 activated polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; MBP, myelin basic protein. only p38a and p38g. The lack of activation of p38b2 by MKK3 This paper is available on line at http://www.jbc.org 1741 This is an Open Access article under the CC BY license. 1742 Molecular Cloning of Human p38b2 MAP Kinase was due to its inability to phosphorylate p38b2. These data demonstrate that the p38b2 MAP kinase is selectively acti- vated by MKK6. We conclude that different p38 MAP kinase isoforms are regulated by overlapping and distinct signal transduction pathways. EXPERIMENTAL PROCEDURES Materials—IL-1a and TNF-a were from Genzyme Corp. MBP was from Sigma. [g- P]ATP was obtained from Amersham Corp. GST-c-Jun (43), GST-ATF2 (44), GST-Elk-1 (45), GST-MKK3 (30), GST-MKK4 (30), and GST-MKK6 (11) fusion proteins have been described. Recom- binant Mapkap-K2 was obtained from Upstate Biotechnology Inc. The drug SB203580 was provided by Dr. M. S.-S. Su, Vertex Pharmaceuti- cals Inc. Mammalian expression vectors for p38a, MKK3, MKK4, and MKK6 have been described (11, 12, 30). The plasmid pCMV-Flag-Elk-1 was provided by Dr. A. J. Whitmarsh (University of Massachusetts Medical School) and Dr. A. Sharrocks (Newcastle University Medical School). The plasmid pCDNA3-Flag p38b1 (23) was provided by Dr. J. Han (The Scripps Research Institute). The p38g cDNA (24, 26, 27) was prepared by RT-PCR amplification using rat skeletal muscle mRNA as the template. A human p38a cDNA was isolated from a fetal brain library by RT-PCR using primers specific for the 59- and 39-untranslated regions. This cDNA was subcloned in the vector pCDNA3 (Invitrogen Inc.) and sequenced. The human p38a MAP kinase cDNA was labeled with [ P]phosphate by random priming and was used to screen a human fetal brain library cloned in the phage lZAPII (Stratagene). Two clones related to p38b1 (designated p38b2) were isolated and sequenced. The mammalian expression vector and the bacterial expression vec- tor for p38a, p38b1, p38b2, and p38g MAP kinase were constructed by subcloning the cDNA in the plasmids pCDNA3 (Invitrogen) and pG- STag (46), respectively. The Flag epitope (-Asp-Tyr-Lys-Asp-Asp-Asp- Asp-Lys-; Immunex Corp.) was inserted between codons 1 and 2 of the p38 MAP kinases by insertional overlapping PCR (47). The sequence of each plasmid was confirmed by automated sequencing using an Applied Biosystems model 373A machine. The GST-p38 fusion proteins were purified by affinity chromatography using glutathione-agarose (48). Tissue Culture—Chinese hamster ovary (CHO) cells were main- tained in Ham’s F-12 medium supplemented with 5% fetal bovine FIG.1. Comparison of the primary sequence of p38b2 MAP serum. HeLa and COS-1 cells were maintained in Dulbecco’s modified kinase with the p38a, p38b1, and p38g MAP kinase isoforms. The primary sequence of human p38b2 MAP kinase was deduced from the Eagle’s medium supplemented with 10% fetal bovine serum (Life Tech- sequence of cDNA clones. The sequence of p38b2 is aligned to the p38a, nologies, Inc.). Plasmid DNA (0.1–1.0 mg) was transfected by the Lipo- p38b1, and p38g MAP kinase isoforms. Residues that are identical to fectAMINE reagent (Life Technologies, Inc.) according to the manufactur- p38a MAP kinase are indicated with a period (.). The sites of activating ers’ recommendations. The cells were harvested after 48 h of incubation. phosphorylation (Thr and Tyr) are indicated with asterisks. The cDNA Immunoprecipitation and Protein Kinase Assays—Cells were solubi- sequence of the human p38b2 MAP kinase has been deposited in Gen- lized with buffer A (20 mM Tris (pH 7.5), 10% glycerol, 1% Triton X-100, TM Bank with accession no. AF031135. The sequences of the p38a (4, 12, 0.137 M NaCl, 25 mM b-glycerophosphate, 2 mM EDTA, 0.5 mM dithio- 16, 17, 22), p38b (23), and p38g (24 –27) MAP kinase isoforms have been threitol, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 10 reported previously. mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). The extracts were centrifuged at 15,000 3 g (15 min at 4 °C). Epitope-tagged protein kinases were immunoprecipitated by incubation for2hat4 °C with the b-galactosidase expression vector pCH110 (Pharmacia-LKB). The effect M2 Flag monoclonal antibody (IBI-Kodak) bound to protein G-Sepha- of co-transfection with 100 ng of expression vectors for p38 MAP kinase, rose (Pharmacia-LKB Biotechnology, Inc). The immunoprecipitates MKK3, MKK4, MKK6, or the empty expression vector was examined. were washed twice with buffer A and twice with kinase buffer (25 mM The cells were harvested 48 h post-transfection. The b-galactosidase HEPES, pH 7.4, 25 mM b-glycerophosphate, 25 mM MgCl , 0.5 mM and luciferase activity in the cell lysates was measured as described dithiothreitol, 0.1 mM sodium orthovanadate). previously (45). Protein kinase assays were performed using recombinant protein kinases and protein kinase immunoprecipitates. The reactions were RESULTS initiated by addition of 1 mg of substrate proteins and 50 mM [g- P]ATP Molecular Cloning of p38b2 MAP Kinase—To identify novel (10 Ci/mmol) in a final volume of 40 ml of kinase buffer. The phospho- members of the human p38 MAP kinase group, we used a rylation reaction was linear with time for at least 40 min. The reactions human p38a MAP kinase cDNA as a probe to screen a human were terminated after 30 min at 30 °C by addition of Laemmli sample buffer. Phosphorylation of the substrate proteins was examined after fetal brain cDNA library. Two cDNA clones related to p38a SDS-polyacrylamide gel electrophoresis (PAGE) by autoradiography MAP kinase were identified. Partial sequence analysis demon- and PhosphorImager analysis. strated that these clones were identical to the p38b MAP ki- Western Blot Analysis—Proteins were fractionated by SDS-PAGE, nase reported by Jiang et al. (23). However, following comple- electrophoretically transferred to an Immobilon-P membrane (Millipore tion of the sequence analysis, it was apparent that the novel Inc.), and probed with the M2 monoclonal antibody to the Flag epitope cDNAs differed from p38b MAP kinase. A deletion of 24 base (IBI-Kodak), a monoclonal antibody to MKK4 (Pharmingen), and an affinity-purified polyclonal antibody to phospho-Ser-383 Elk-1 (New pairs was detected in the sequence of the novel clones compared England Biolabs). Immunocomplexes were detected using chemilumi- with p38b MAP kinase. This gap results in the deletion of an nescence (Lumiglo; Kirkegaard & Perry Laboratories). 8-amino acid insertion present in p38b MAP kinase (23). We Measurement of Reporter Gene Expression—Transfection assays designate the novel sequence as p38b2 and the previously were performed using CHO cells and the LipofectAMINE method (Life reported sequence p38b1 (Fig. 1). Technologies, Inc.). The cells were co-transfected with 0.2 mg of pGAL4- It is possible that the 8-amino acid deletion was the result of Elk-1 (45) and 0.2 mg of the reporter plasmid pG5E1bLuc (49). Trans- fection efficiency was normalized by co-transfection of the cells with the alternative splicing. To test this hypothesis, we performed RT- Molecular Cloning of Human p38b2 MAP Kinase 1743 FIG.3. Substrate specificity of p38 MAP kinases. A, epitope- tagged p38 MAP kinases were transfected in COS cells. The cells were then irradiated with 80 J of UV-C per m (1) or left untreated (2). The p38 MAP kinases were immunopurified and substrate phosphorylation by each p38 MAP kinase was examined in an immuncomplex protein kinase assay. The level of expression of the epitope-tagged p38 MAP FIG.2. The p38b2 protein kinase is a novel MAP kinase. A, kinases was examined by Western blot analysis using the M2 mono- recombinant GST-p38b1 and p38b2 were incubated with [g P]ATP clonal antibody (lower panel). B, the phosphorylation of ATF2 was and buffer (2) or GST-ATF2 (ATF2)(1). The phosphorylation reaction examined using immunopurified p38 MAP kinases prepared from was terminated by addition of Laemmli sample buffer, and the phos- COS-7 cells exposed to UV radiation. The effect of replacement of the phorylated proteins were detected after SDS-PAGE by autoradiogra- phosphorylation sites Thr-69 and Thr-71 with Ala residues is presented. phy. The ATF2, p38b1, and p38b2 are indicated with arrowheads. B, COS-7 cells expressing epitope-tagged p38a, p38b1, p38b2, and p38g The absence of protein kinase activity detected for p38b1 in were exposed to 80 J of UV-C per m (1) and compared with control cells (2). The p38 MAP kinases were isolated by immunoprecipitation and vitro may not represent the activity of this protein kinase in used for protein kinase assays with ATF2 as the substrate (upper vivo. We therefore tested the activity of p38b1 and p38b2 in panel). The phosphorylation reaction was initiated by the addition of vivo. Equal amounts of these p38 MAP kinase isoforms were [g- P]ATP and ATF2. The level of expression of the epitope-tagged p38 expressed in COS-7 cells (Fig. 2B). Exposure to UV-C radiation MAP kinases was examined by Western blot analysis using the M2 monoclonal antibody (lower panel). C, epitope-tagged p38a, p38b1, caused increased protein kinase activity of p38b2, but not p38b2, and p38g were immunoprecipitated from cells co-transfected p38b1 (Fig. 2B). Control experiments demonstrated that UV- with empty vector (2) or activated MKK6 (1) and used for kinase activated p38b2 was less active than UV-activated p38a or assays with ATF2 as a substrate (upper panel). The level of expression p38g (Fig. 2B). of the epitope-tagged MKK6 and the p38 MAP kinases was examined by Western blot analysis using the M2 monoclonal antibody (lower panel). The absence of p38b1 activity and the low level of p38b2 activity compared with p38a and p38g may indicate that UV PCR using primers that span the deletion (59-TCCATCGAG- radiation is a poor activator of p38b1 and p38b2 protein kinase GACTTCAGCGAAGTG-39 and 59-GCCTGGCGCGCCAGCCC- activity. We therefore examined the activity of the p38b MAP GAAATC-39). Sequence analysis of the products of amplifica- kinase isoforms in co-transfection experiments with constitu- tion of human brain mRNA led to the identification of p38b2, tively activated MKK6, a strong activator of p38 MAP kinases but not p38b1. These data demonstrate that in human brain (11). These experiments demonstrated that the activation of p38b2 is the major p38b isoform. It is possible that p38b1 may p38b2 MAP kinase was similar to the activation of p38a and be expressed in other tissues. p38g MAP kinases (Fig. 2C). In contrast, the p38b1 MAP Biochemical Characterization of p38b2 MAP Kinase Activity kinase isoform was inactive in this assay (Fig. 2C). in Vivo and in Vitro—The phosphorylation of ATF2 by p38a Substrate Phosphorylation by p38b2 MAP Kinase—We im- MAP kinase has been studied in detail (11, 12). We therefore munopurified p38a, p38b2, and p38g MAP kinases from control tested whether ATF2 was a substrate for p38b2. In vitro pro- and UV-irradiated cells. The amount of each Flag-tagged p38 tein kinase assays using recombinant p38b2 demonstrated MAP kinase was examined by immunoblot analysis using the that this protein kinase did autophosphorylate (Fig. 2A). In M2 monoclonal antibody. An equal amount of each p38 MAP contrast, control studies using recombinant p38b1 did not dem- kinase isoform was used for in vitro protein kinase assays using onstrate autophosphorylation (Fig. 2A). Addition of ATF2 re- different substrates. This analysis demonstrated that ATF2, sulted in phosphorylation by p38b2, but not by p38b1 (Fig. 2A). Elk-1, and MBP were phosphorylated by p38b2 (Fig. 3A). These data suggest that p38b2 is a more active protein kinase ATF2, Elk-1, and MBP were also phosphorylated by p38a and than p38b1. p38g MAP kinases (Fig. 3A). The extent of substrate phospho- 1744 Molecular Cloning of Human p38b2 MAP Kinase rylation by p38b2 was less than p38a and p38g. However, the fold-activation of p38b2 activity was similar to that detected for p38a and p38g (Fig. 3A). These data indicate that the substrate specificity of these p38 MAP kinase isoforms was similar. To further examine the substrate specificity of p38b2 MAP kinase, we examined the phosphorylation of two other sub- strates for stress-activated MAP kinases. First, the transcrip- tion factor c-Jun, which is phosphorylated by JNK (3). We found that c-Jun was not phosphorylated by any of the p38 MAP kinase isoforms tested (data not shown). In a second series of experiments, we examined the phosphorylation of the protein kinase Mapkap-K2, which is reported to be a substrate for p38 MAP kinase (16, 17). These studies indicated that p38a MAP kinase, but not p38b2orp38g, phosphorylated Map- kap-K2 (Fig. 3A). Thus, the p38b2 MAP kinase substrate spec- ificity differs from p38a MAP kinase. The results of substrate analysis indicate that the substrate specificity of p38b2 MAP kinase was similar to p38g (Fig. 3A). However, this analysis of substrate phosphorylation does not take account of the sites of phosphorylation of each protein. We therefore performed more detailed analysis of p38b2 MAP ki- nase activity using the transcription factor ATF2 as the sub- strate. ATF2 was found to be a substrate for p38a, p38b2, and p38g MAP kinase (Fig. 3A). We have previously reported that FIG.4. The p38b2 MAP kinase is inhibited by SB203580. The effect of SB203580 on p38 MAP kinases was tested in an in vitro protein p38a MAP kinase phosphorylates ATF2 on Thr-69 and Thr-71 kinase assay. Recombinant p38a, p38b2, and p38g MAP kinases were (12). Mutational analysis confirmed this conclusion. Replace- incubated without SB203580 (lanes 1 and 2) or with 0.1, 1, and 10 mM ment of Thr-69 or Thr-71 with Ala caused decreased phospho- of the drug (lanes 3, 4, and 5, respectively) in the presence of [g- P]ATP rylation of ATF2, while replacement of both phosphorylation (lanes 1–5) and ATF2 as a substrate (lanes 2–5). The reaction was sites with Ala eliminated ATF2 phosphorylation by p38a MAP terminated by the addition of Laemmli sample buffer, and the phos- phorylated proteins were detected after SDS-PAGE by autoradiogra- kinase (Fig. 3B). In contrast, experiments using p38g MAP phy. The rate of phosphorylation was quantitated by PhosphorImager kinase demonstrated that the replacement of Thr-71 with Ala analysis and is presented as p38 MAP kinase activity relative to the caused a marked decrease in ATF2 phosphorylation, while kinase activity in the absence of SB203580 (1.0). replacement of Thr-69 with Ala increased ATF2 phosphoryla- tion (Fig. 3B). These data suggest that either Thr-71 is the effects of pyridinyl imidazole derivatives on cultured cells (4, 5). major site of ATF2 phosphorylation by p38g MAP kinase, or The p38b2 MAP kinase is therefore likely to be a physiologi- that the phosphorylation of Thr-71 precedes the phosphoryla- cally relevant mediator of the p38 MAP kinase signal trans- tion of other sites by p38g MAP kinase. Comparative studies duction pathway. using p38b2 MAP kinase demonstrate that this MAP kinase The p38b2 MAP Kinase Is Activated by Proinflammatory isoform differs from both p38a and p38g MAP kinase isoforms. Cytokines and Environmental Stress—The p38a and p38g MAP Replacement of Thr-69 with Ala caused a small decrease in kinases are regulated by numerous extracellular stimuli, in- ATF2 phosphorylation, replacement of Thr-71 with Ala caused cluding proinflammatory cytokines and environmental stress a larger decrease in ATF2 phosphorylation, and replacement of (38). We compared the regulation of p38b2 MAP kinase with both residues caused a marked reduction, but not the elimina- the p38a and p38g MAP kinases. HeLa cells were transfected tion, of ATF2 phosphorylation by p38b2 (Fig. 3B). These data with epitope-tagged p38a, p38b2, and p38g MAP kinases and suggest that both Thr-69 and Thr-71 are phosphorylated by exposed to different extracellular stimuli. The activity of each p38b2 MAP kinase. However, the phosphorylation of ATF2 by p38 MAP kinase isoform was detected by measurement of p38b2 MAP kinase following replacement of both Thr-69 and protein kinase activity in an immune complex kinase assay Thr-71 with Ala residues indicate that p38b2 phosphorylates a using ATF2 as the substrate. The proinflammatory cytokines novel site on ATF2. Together, these data demonstrate that the TNF-a and IL-1a, environmental stress (UV irradiation and substrate specificity of p38b2 MAP kinase differs from the osmotic shock), and treatment with anisomycin (an inhibitor of p38a and p38g MAP kinase isoforms. protein synthesis) caused a marked increase in the activity of Effect of a Pyridinyl Imidazole Drug on p38b2 MAP Kinase p38b2 MAP kinase (Fig. 5). The strongest activation of p38b2 Activity—We examined the effect of the pyridinyl imidazole was caused by the exposure of cells to UV radiation. Although derivative SB203580 (4, 5) on the protein kinase activity of ATF2 phosphorylation by p38b2 was consistently less than p38b2 MAP kinase. This drug has previously been shown to that observed in kinase assays with p38a or p38g, the fold- inhibit p38a MAP kinase activity (4, 5). Here we demonstrate increase in protein kinase activity caused by UV radiation was that SB203580 inhibits p38b2 MAP kinase activity (Fig. 4). similar for each p38 MAP kinase isoform (Fig. 5). These data The dose response of inhibition of protein kinase activity was demonstrate that p38b2, like p38a and p38g, is activated in similar in experiments using p38a and p38b2 MAP kinase (Fig. vivo by a stress-induced signal transduction pathway. 4). In contrast, p38g MAP kinase was not inhibited by Selective Activation of p38 MAP Kinases by MAP Kinase SB203580. The insensitivity of p38g MAP kinase to inhibition Kinases—The p38 MAP kinases are activated in response to by SB203580 observed in this study differs from the results of extracellular stimuli by dual phosphorylation on Thr and Tyr one previous study (26), but is in agreement with more recent by the MAP kinases kinases MKK3 (30), MKK4 (30, 34), and studies by other investigators (25). As the p38a and p38b2 MKK6 (11, 31–33). We therefore tested the effect of MKK3, isoforms demonstrate similar inhibition by SB203580, both of MKK4, and MKK6 on p38b2 MAP kinase activity in co-trans- these MAP kinases could account for the previously reported fection assays in vivo. Control experiments were performed Molecular Cloning of Human p38b2 MAP Kinase 1745 FIG.5. The p38b2 MAP kinase is activated by pro-inflamma- tory cytokines and environmental stress. The activity of epitope- tagged p38a, p38b2 and p38g MAP kinases was measured in immune complex protein kinase assays using [g- P]ATP and ATF2 as substrate. The effect of treatment of Hela cells (30 min) with 10 ng/ml TNF-a,10 ng/ml IL-1a, 300 mM sorbitol, 10 mg/ml anisomycin, and 80 J of UV-C per m was examined. The phosphorylated ATF2 was detected by au- toradiography (15 min). To detect the lower level of p38b2 activity, a longer autoradiographic exposure (45 min) of the phosphorylated ATF2 is also presented. The rate of phosphorylation was quantitated by FIG.6. Selective activation of p38 MAP kinase isoforms by PhosphorImager analysis and is presented as the percentage of p38 MAP kinase kinases in vivo. The ability of MKK3 (panel A), MKK4 MAP kinase activity relative to cells treated with UV radiation (100%). (panel B), and MKK6 (panel C) to activate p38a, p38b2, and p38g was tested in co-transfection assays. COS-7 cells were transfected with epitope-tagged p38a, p38b2, or p38g together with an empty vector using p38a and p38g MAP kinases. MKK3 caused strong acti- (Control) or an expression vector encoding epitope-tagged constitutively vation of p38a, a lower level of activation of p38g, and did not activated MKK3, MKK4, and MKK6. The p38 MAP kinase activity was activate p38b2 (Fig. 6A). Similarly, MKK4 activated p38a measured in an immune complex kinase assay using ATF2 as the substrate. The level of expression of the p38 MAP kinases and the MAP strongly, weakly activated p38g, and did not activate p38b2 kinase kinases was examined by Western blot analysis. (Fig. 6B). In contrast, MKK6 caused strong activation of p38a, p38b2, and p38g MAP kinases (Fig. 6C). The effect of MKK3 cause potent stimulation of co-transfected p38 MAP kinase and MKK4 to activate p38a and p38g, but not p38b2, indicates activity (11). Activated MKK3 can therefore be used as a tool to that the regulation of p38b2 MAP kinase differs from the other test the contribution of specific p38 MAP kinases isoforms on p38 MAP kinases. The selective effect of MAP kinase kinases to cellular responses. Co-transfection assays demonstrated that regulate p38b2 MAP kinase activity suggests that extracellular activated MKK3 did increase Elk-1-dependent luciferase gene stimuli may selectively regulate p38 MAP kinase isoforms. expression when co-transfected with p38 a and p38g MAP ki- The p38b2 MAP Kinase Is a Substrate for MKK6, but Not nases (Fig. 8, A and C). In contrast, p38b2 MAP kinase did not MKK3—The effect of MKK3 to activate p38a and p38g MAP increase MKK3-stimulated Elk-1 transcriptional activity (Fig. kinases, but not p38b2, could be accounted for by many mech- 8B). Control experiments using activated MKK6 demonstrated anisms. One possibility is that p38b2 is not a substrate for that p38 a, p38b2, and p38g MAP kinases increased MKK6- MKK3. To test this hypothesis, we examined the phosphoryla- stimulated Elk-1 transcriptional activity (Fig. 8, A–C). Consist- tion of p38 MAP kinase isoforms by MKK3 and MKK6 (Fig. 7). ent with these data, MKK6 caused a similar level of phospho- In vitro protein kinase assays demonstrated the autophospho- rylation of Elk-1 by each of the three p38 MAP kinases in vitro rylation of MKK3, but not MKK6, as described previously (11, (Fig. 8D) and in vivo (Fig. 8E). Similar data were obtained in 30). MKK6 caused strong phosphorylation of p38a and p38b2 experiments using the transcription factor ATF2 as a p38 MAP MAP kinase, and caused a lower level of phosphorylation of kinase substrate (data not shown). p38g MAP kinase (Fig. 7B). In contrast, MKK3 caused a sim- Together, these data indicate that MKK6 can couple to p38a, ilar level of phosphorylation of p38a and p38g MAP kinases, p38b2, and p38g MAP kinases to initiate a biological response, but no phosphorylation of p38b2 (Fig. 7A). These data demon- while MKK3 can couple only to p38a and p38g MAP kinases. strate that MKK3 and MKK6 differentially phosphorylate p38 These data are consistent with the potent activation of p38b2 MAP kinase isoforms. Furthermore, these data indicate that by MKK6 and the ineffective activation of p38b2 by MKK3 in p38b2 MAP kinase is a substrate for MKK6, but not MKK3. vitro and in vivo (Figs. 6 and 7). Transcriptional Regulation by the MKK6-p38b2 MAP Kinase DISCUSSION Signaling Pathway—The constitutively active form of MKK3 does not activate endogenous p38 MAP kinase in transient The stress-activated MAP kinases include the JNK and p38 transfection assays (11). In contrast, activated MKK3 does groups (3). The JNK group consists of 10 members that are 1746 Molecular Cloning of Human p38b2 MAP Kinase insertion present in p38b1 may be derived by post-transcrip- tional processing of the p38b2 transcript. Further studies are required to resolve this issue. Our studies of the origin of p38b1 have been limited because we have been unable to detect p38b1 by RT-PCR analysis of human mRNA. No difficulty was experienced in the detection of p38b2. We interpret these data to indicate that p38b2isthe major p38b MAP kinase isoform that is expressed in many human tissues. The expression of the p38b1 isoform may be restricted to specific tissues. The p38b2 MAP Kinase Is a Stress-activated Protein Ki- nase—The p38b2 MAP kinase, like the p38a and p38g MAP kinase isoforms, is activated by treatment of cells with proin- flammatory cytokines (e.g. TNF and IL-1) or by exposure of cells to environmental stress (e.g. UV radiation and osmotic shock) (Fig. 5). The p38b2 MAP kinase shares overlapping, but distinct, substrate specificity with the p38a and p38g MAP kinase isoforms (Fig. 2A). The p38b2 MAP kinase was not observed to phosphorylate c-Myc, IkB, Max, Smad1, c-Fos, and NFAT (data not shown). However, p38b2 was found to phos- phorylate the p38a and p38g substrates ATF2, Elk-1, and MBP FIG.7. Selective phosphorylation of p38 MAP kinase isoforms (Fig. 3A). Mutational analysis of the sites of ATF2 phosphoryl- by MAP kinase kinases. The effect of recombinant MKK3 (panel A) ation demonstrated that the substrate specificity of p38b2 dif- and MKK6 (panel B) to phosphorylate p38 MAP kinases was examined fers from p38a and p38g (Fig. 3B). in an in vitro protein kinase assay using recombinant p38a, p38b2, and p38g MAP kinases. The reactions were initiated by addition of Intriguingly, we have not been able to detect any protein [g- P]ATP. The reaction products were examined by SDS-PAGE and kinase activity in experiments using the p38b1 MAP kinase. autoradiography. The p38 MAP kinase isoforms are indicated by aster- Similar negative data were obtained in vitro and in vivo (Fig. isks (*). The MKK3 and MKK6 are indicated by arrowheads. 2). The lack of activity of p38b1 is in contradiction with studies by Jiang et al. (23) who reported that p38b1 is a constitutively derived by alternative splicing of three genes (50). These JNK activated kinase that preferentially phosphorylates ATF2. The isoforms differ in their tissue distribution and in their interac- reason for this discrepancy is unclear, as we did not detect any tion with substrate proteins (50). It has therefore been pro- phosphorylation of ATF2 by recombinant p38b1 in vitro (Fig. posed that individual JNK isoforms may mediate distinct phys- 1A), nor have we been able to activate transfected p38b1byUV iological responses (50). Similarly, the p38 group of stress- irradiation (Fig. 2B) or by cotransfection with activated MKK6 activated MAP kinases consists of multiple isoforms (1). These (Fig. 2C). Further studies are required to resolve this isoforms include p38a (4, 12, 16, 17, 22), p38b (23), and p38g discrepancy. (24 –27). Recent studies indicate the presence of a fourth p38 Northern blot analysis of p38a and p38b MAP kinases dem- MAP kinase isoform, p38d (28, 29). In addition, alternatively onstrates that the tissue distribution of these isoforms is very spliced forms of p38a MAP kinase have been described (4, 51). similar (23). Consequently, the inhibition of both enzymes by The existence of multiple p38 MAP kinase isoforms provides SB203580 (Fig. 4) suggests that some of the physiological func- the potential for the generation of stimulus-specific and cell tions attributed to p38a, based on the effects of pyridinyl im- type-specific responses to activation of the p38 MAP kinase idazole drugs, could be mediated by p38b2 MAP kinase. There- signaling pathway. The identification of p38 MAP kinase iso- fore, p38b2 may contribute to the regulation of the production forms and their mechanism of activation by MAP kinase ki- of TNF and IL-1 by monocytes in response to lipopolysaccha- nases represents one step that is required for understanding ride (4), the induction of the IL-6 gene expression by TNF in the physiological role of p38 MAP kinases in mammalian cells. fibroblasts (6), or any other cellular responses blocked by Here we describe a novel p38 MAP kinase isoform (p38b2) that SB203580 (5). is selectively activated by the MAP kinase kinase MKK6. MKK3 and MKK6 Differentially Activate p38 MAP Kinase The p38b2 Protein Kinase Is a Novel MAP Kinase—We re- Isoforms—The p38 MAP kinases kinases MKK3, MKK4, and port the molecular cloning of p38b2 MAP kinase, a novel hu- MKK6 selectively activate the p38a, p38b2, and p38g MAP man stress-activated protein kinase. This enzyme is most sim- kinase isoforms (Fig. 9). MKK6 activates p38a, p38b2, and ilar to the previously characterized p38b MAP kinase (p38b1) p38g, while MKK3 and MKK4 only activate p38a and p38g (23) and may be derived from the same gene by alternative (Fig. 6). The lack of activation of p38b2 by MKK3 is explained splicing. The p38b2 MAP kinase contains a 24-base pair dele- by its inability to phosphorylate p38b2, while MKK6 phospho- tion within the coding region of p38b1 MAP kinase. This dele- rylates all three isoforms (Fig. 7). The specificity of MKK3 and tion represents a significant difference between these p38b MKK6 to differentially activate these p38 MAP kinase isoforms MAP kinase isoforms. The p38b1 MAP kinase contains an was confirmed by analysis of the effects of MKK3 and MKK6 on 8-amino acid insertion in the kinase domain that is absent in Elk-1-dependent gene expression (Fig. 8). p38b2 MAP kinase (Fig. 1). It is most likely that p38b1 and p38b2 MAP kinases repre- The observation that MAP kinase kinases differentially reg- ulate p38 MAP kinase isoforms has important implications for sent alternatively spliced forms of the same gene. However, sequence analysis of a p38b2 MAP kinase genomic clone dem- the specificity of signal transduction mechanisms. Stimuli that selectively activate MAP kinases kinases would lead to the onstrated that the site of the p38b1 insertion was present within an exon (not at an exon boundary) and that the 24 base activation of different groups of p38 MAP kinase isoforms. This pair p38b1 insertion sequence was not detected in the genomic selective activation of MAP kinases provides a mechanism for clone (data not shown). These data suggest that p38b1 and the generation of stimulus-specific responses of cells to their p38b2 may be encoded by different genes. Alternatively, the environment. However, this mechanism does require that spe- Molecular Cloning of Human p38b2 MAP Kinase 1747 FIG.9. The p38 MAP kinase kinases MKK3, MKK4, and MKK6 cause selective activation of p38 MAP kinase isoforms. The MAP kinase kinase MKK6 phosphorylates and strongly activates p38a, p38b2, and p38g. MKK3 activates p38a and p38g, but not p38b2. The MAP kinase kinase MKK4 activates p38a and does not activate p38b2. MKK4 is also a weak activator p38g. The differential activation of these p38 MAP kinase isoforms may lead to cell type-specific and stimulus- specific cellular responses. cific stimuli cause the differential activation of MAP kinase kinases. It is established that nonrelated MAP kinase kinases are selectively activated in response to the treatment of cells with different stimuli (3). For example, MEK1 is activated by phor- bol ester and MKK3 is activated by UV radiation (30). How- ever, detailed comparative studies of related MAP kinases ki- nases have not yet been completed. It is therefore not clear whether the selective activation of related MAP kinase kinases (e.g. MKK3 and MKK6) is a common event or whether it is unusual. Further studies are required to provide an answer to this question. A precedent for the selective activation of related MAP ki- nases kinases has been established by previous studies of the ERK pathway (52). It has been shown that a proline-rich region in MEK1 and MEK2, the upstream activators of ERK, is nec- essary for recognition and activation by Raf family kinases (53). Phosphorylation of Thr-292 in the proline-rich region of MEK1 regulates the kinetics of inactivation of MEK1 following stim- ulation by growth factors (53). MEK2 lacks this regulatory phosphorylation site and is inactivated more rapidly than MEK1 (53). This regulatory phosphorylation of the proline-rich region of MEK1 provides a mechanism for the differential activation of MEK1 and MEK2 by serum and other stimuli (53). Recent studies have demonstrated the differential regulation of the p38 MAP kinase kinases MKK3 and MKK6. Treatment of Jurkat cells with FAS ligand causes caspase-dependent ac- tivation of the p38 MAP kinase signal transduction pathway (54). FAS-ligation activates MKK6, but not MKK3, in these cells with kinetics which correlated with the onset of FAS- induced apoptosis (55). Constitutively activated MKK6 in- creased the number of apoptotic cells, while dominant-negative MKK6 increased the number of surviving cells following FAS cross-linking (55). These data indicate that FAS ligation selec- tively activates the MKK6, but not MKK3, signaling pathway. These data suggest that MKK3 and MKK6 can be specifically activated by extracellular stimuli and that they have distinct roles. It is likely that such selective activation of p38 MAP kinase kinase isoforms occurs in response to other stimuli and on Elk-1 phosphorylation in vitro. Elk-1 phosphorylation was examined using epitope-tagged p38 MAP kinase isoforms immunoprecipitated from COS cells co-transfected with empty vector (2) or activated MKK6 FIG.8. Regulation of gene expression by p38 MAP kinase iso- (1). The level of expression of epitope-tagged p38 MAP kinases and forms. A–C, effect of p38 MAP kinase isoforms on Elk-1 transcriptional MKK6 was examined by Western blot analysis. E, effect of p38 MAP activity. Elk-1-dependent gene expression was examined in CHO cells kinase isoforms on Elk-1 phosphorylation in vivo. COS cells were co- co-transfected with the b-galactosidase expression vector pCH110, the transfected with Elk-1 and p38 MAP kinase isoforms with empty vector reporter plasmid pG5E1bLuc, and an expression vector for the GAL4 (2) or activated MKK6 (1). The level of expression of epitope-tagged DNA binding domain (residue 1–147) fused to the transcription factor p38 MAP kinases, MKK6, and Elk-1 was examined by Western blot Elk-1 (residue 307– 428). Transfection efficiency was monitored by analysis. The phosphorylation of Elk-1 was examined by Western blots measurement of b-galactosidase expression. The relative luciferase ac- probed with an antibody that binds Elk-1 phosphorylated at Ser-383. tivity detected following co-transfection of the empty vector, activated Previous studies have established that the phosphorylation of Elk-1 on MKK3, or activated MKK6 together with p38a (panel A), p38b2(panel Ser-383 contributes to increased transcriptional activity caused by p38 B), or p38g (panel C) is presented. D, effect of p38 MAP kinase isoforms MAP kinase (56, 57). 1748 Molecular Cloning of Human p38b2 MAP Kinase 24. Lechner, C., Zahalka, M. A., Giot, J.-F., Moller, N. P., and Ullrich, A. (1996) in other cell types. Proc. Natl. Acad. Sci. U. S. A. 93, 4355– 4359 Conclusions—The cellular response to treatment with proin- 25. Cuenda, A., Cohen, P., Buee-Scherrer, V., and Goedert, M. 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Biol. 17, 2360 –2371 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Selective Activation of p38 Mitogen-activated Protein (MAP) Kinase Isoforms by the MAP Kinase Kinases MKK3 and MKK6

Enslen, Hervé; Raingeaud, Joël; Davis, Roger J.
Journal of Biological Chemistry – Jan 1, 1998
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 3, Issue of January 16, pp. 1741–1748, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Selective Activation of p38 Mitogen-activated Protein (MAP) Kinase Isoforms by the MAP Kinase Kinases MKK3 and MKK6* (Received for publication, July 28, 1997, and in revised form, October 27, 1997) Herve ´ Enslen, Joe ¨ l Raingeaud, and Roger J. Davis‡ From the Howard Hughes Medical Institute, Program in Molecular Medicine, and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 The cellular response to treatment with proinflamma- and IL-7 (7), and the stress-induced transcription of c-jun and tory cytokines or exposure to environmental stress is c-fos in fibroblasts (8). The targets of p38 MAP kinase that mediated, in part, by the p38 group of mitogen-activated mediate these responses are poorly characterized. However, protein (MAP) kinases. We report the molecular cloning biochemical studies indicate that p38 MAP kinase signaling b2. This p38 of a novel isoform of p38 MAP kinase, p38 pathway activates the transcription factors CREB and ATF1 a, is inhibited by the pyridinyl im- MAP kinase, like p38 (9, 10), ATF2 (11, 12), CHOP (13), and MEF-2C (14). The p38 idazole drug SB203580. The p38 MAP kinase kinase MAP kinase also activates other protein kinases, such as Map- a, MKK6 is identified as a common activator of p38 kap-2 (15–17), Mapkap-3 (18, 19), and Mnk1/2 (20, 21). b2, and p38g MAP kinase isoforms, while MKK3 ac- p38 The p38 MAP kinase group includes the isoforms p38a (4, 12, a and p38g MAP kinase isoforms. The tivates only p38 16, 17, 22), p38b (23), and p38g (24 –27). Recent studies indi- MKK3 and MKK6 signal transduction pathways are cate the presence of a fourth p38 MAP kinase isoform, p38d (28, therefore coupled to distinct, but overlapping, groups of 29). These p38 MAP kinases are widely expressed in many p38 MAP kinases. tissues and are activated by dual phosphorylation on Thr and Tyr within the motif Thr-Gly-Tyr located in kinase subdomain VIII (12). This phosphorylation is mediated by a protein kinase Mitogen-activated protein (MAP) kinases are proline-di- cascade (1). Components of this signaling pathway include the rected protein kinases that mediate the effects of numerous MAP kinase kinases MKK3 (30) and MKK6 (11, 31–33). It is extracellular stimuli on a wide array of biological processes, also possible that MKK4 contributes to the activation of p38 such as cellular proliferation, differentiation, and death (1). MAP kinase. In vitro studies demonstrate that MKK4 activates Three groups of mammalian MAP kinases have been studied in both JNK and p38 MAP kinases (30, 34). However, the role of detail: the extracellular signal-regulated kinases (ERK) (2), the MKK4 as an activator of p38 MAP kinase in vivo is unclear c-Jun NH -terminal kinases (JNK) (3), and the p38 MAP ki- (35). nases (3). The ERKs are robustly activated by growth factors The activation of MKK3 and MKK6 is regulated by phospho- and phorbol ester, but are only weakly activated by cytokines rylation on Ser and Thr residues within subdomain VIII by and environmental stress. In contrast, JNK and p38 MAP MAP kinase kinase kinases (MKKK) (1). Further studies are kinases are strongly activated by cytokines and environmental required to define the function and specificity of MKKKs that stress, but are poorly activated by growth factors and phorbol ester. cause activation of the p38 MAP kinase pathway. However, one candidate MKKK for the p38 MAP kinase signaling pathway is Recently, progress toward understanding the physiological role of the p38 MAP kinases has been achieved through the use TAK1, which has been reported to activate MKK3 and MKK6 of drugs that bind p38 MAP kinase (4, 5). These drugs are (36 –38). Other MKKKs, which activate both JNK and p38 pyridinyl imidazole derivatives that inhibit p38 MAP kinase MAP kinases, include ASK-1 (39) and the mixed-lineage kinase activity (4, 5). Studies using these drugs indicate that p38 MAP MLK-3 (40 – 42). Other MKKKs that activate the JNK signal- kinase is required for lipopolysaccharide-induced production of ing pathway, for example MEKK1, do not cause activation of IL-1 and TNF in monocytes (4), the induction of IL-6 and p38 MAP kinase (30, 34). granulocyte-macrophage/colony-stimulating factor transcrip- The expression of multiple p38 MAP kinase isoforms in tion by TNF (6), the proliferation of T cells in response to IL-2 mammalian tissues suggests that these MAP kinases may dif- fer in their physiological function. These p38 MAP kinases may be coupled to different upstream signaling pathways. This * These studies were supported in part by National Institutes of would enable the activation of specific p38 MAP kinase iso- Health, NCI, Grants CA65861 and CA72009. The costs of publication of forms in response to different stimuli. Alternatively, these p38 this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance MAP kinase isoforms may differ in their substrate specificity. with 18 U.S.C. Section 1734 solely to indicate this fact. Such differences could allow coupling of different p38 MAP The nucleotide sequence(s) reported in this paper has been submitted TM kinase isoforms to different signal transduction targets. to the GenBank /EBI Data Bank with accession number(s) AF031135. ‡ Investigator of the Howard Hughes Medical Institute. To whom The purpose of this study was to examine the p38b MAP correspondence should be addressed: Howard Hughes Medical Insti- kinase signal transduction pathway. We find that p38b MAP tute, Program in Molecular Medicine, University of Massachusetts kinase (23) is not a functional protein kinase in vitro or in vivo. Medical School, 373 Plantation St., Worcester, MA 01605. However, a novel p38b MAP kinase isoform (p38b2) that was The abbreviations used are: MAP, mitogen-activated protein; MKK, MAP kinase kinase; MKKK, MAP kinase kinase kinase; ERK, extra- isolated from a human brain cDNA library encoded a functional cellular signal-regulated kinase; JNK, c-Jun NH -terminal kinase; IL, 2 protein kinase. This novel p38 MAP kinase isoform was inhib- interleukin; TNF, tumor necrosis factor; GST, glutathione S-transfer- ited by pyridinyl imidazole drugs. The MAP kinase kinase ase; RT, reverse transcriptase; PCR, polymerase chain reaction; PAGE, MKK6 activated p38a, p38b2, and p38g, while MKK3 activated polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; MBP, myelin basic protein. only p38a and p38g. The lack of activation of p38b2 by MKK3 This paper is available on line at http://www.jbc.org 1741 This is an Open Access article under the CC BY license. 1742 Molecular Cloning of Human p38b2 MAP Kinase was due to its inability to phosphorylate p38b2. These data demonstrate that the p38b2 MAP kinase is selectively acti- vated by MKK6. We conclude that different p38 MAP kinase isoforms are regulated by overlapping and distinct signal transduction pathways. EXPERIMENTAL PROCEDURES Materials—IL-1a and TNF-a were from Genzyme Corp. MBP was from Sigma. [g- P]ATP was obtained from Amersham Corp. GST-c-Jun (43), GST-ATF2 (44), GST-Elk-1 (45), GST-MKK3 (30), GST-MKK4 (30), and GST-MKK6 (11) fusion proteins have been described. Recom- binant Mapkap-K2 was obtained from Upstate Biotechnology Inc. The drug SB203580 was provided by Dr. M. S.-S. Su, Vertex Pharmaceuti- cals Inc. Mammalian expression vectors for p38a, MKK3, MKK4, and MKK6 have been described (11, 12, 30). The plasmid pCMV-Flag-Elk-1 was provided by Dr. A. J. Whitmarsh (University of Massachusetts Medical School) and Dr. A. Sharrocks (Newcastle University Medical School). The plasmid pCDNA3-Flag p38b1 (23) was provided by Dr. J. Han (The Scripps Research Institute). The p38g cDNA (24, 26, 27) was prepared by RT-PCR amplification using rat skeletal muscle mRNA as the template. A human p38a cDNA was isolated from a fetal brain library by RT-PCR using primers specific for the 59- and 39-untranslated regions. This cDNA was subcloned in the vector pCDNA3 (Invitrogen Inc.) and sequenced. The human p38a MAP kinase cDNA was labeled with [ P]phosphate by random priming and was used to screen a human fetal brain library cloned in the phage lZAPII (Stratagene). Two clones related to p38b1 (designated p38b2) were isolated and sequenced. The mammalian expression vector and the bacterial expression vec- tor for p38a, p38b1, p38b2, and p38g MAP kinase were constructed by subcloning the cDNA in the plasmids pCDNA3 (Invitrogen) and pG- STag (46), respectively. The Flag epitope (-Asp-Tyr-Lys-Asp-Asp-Asp- Asp-Lys-; Immunex Corp.) was inserted between codons 1 and 2 of the p38 MAP kinases by insertional overlapping PCR (47). The sequence of each plasmid was confirmed by automated sequencing using an Applied Biosystems model 373A machine. The GST-p38 fusion proteins were purified by affinity chromatography using glutathione-agarose (48). Tissue Culture—Chinese hamster ovary (CHO) cells were main- tained in Ham’s F-12 medium supplemented with 5% fetal bovine FIG.1. Comparison of the primary sequence of p38b2 MAP serum. HeLa and COS-1 cells were maintained in Dulbecco’s modified kinase with the p38a, p38b1, and p38g MAP kinase isoforms. The primary sequence of human p38b2 MAP kinase was deduced from the Eagle’s medium supplemented with 10% fetal bovine serum (Life Tech- sequence of cDNA clones. The sequence of p38b2 is aligned to the p38a, nologies, Inc.). Plasmid DNA (0.1–1.0 mg) was transfected by the Lipo- p38b1, and p38g MAP kinase isoforms. Residues that are identical to fectAMINE reagent (Life Technologies, Inc.) according to the manufactur- p38a MAP kinase are indicated with a period (.). The sites of activating ers’ recommendations. The cells were harvested after 48 h of incubation. phosphorylation (Thr and Tyr) are indicated with asterisks. The cDNA Immunoprecipitation and Protein Kinase Assays—Cells were solubi- sequence of the human p38b2 MAP kinase has been deposited in Gen- lized with buffer A (20 mM Tris (pH 7.5), 10% glycerol, 1% Triton X-100, TM Bank with accession no. AF031135. The sequences of the p38a (4, 12, 0.137 M NaCl, 25 mM b-glycerophosphate, 2 mM EDTA, 0.5 mM dithio- 16, 17, 22), p38b (23), and p38g (24 –27) MAP kinase isoforms have been threitol, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 10 reported previously. mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). The extracts were centrifuged at 15,000 3 g (15 min at 4 °C). Epitope-tagged protein kinases were immunoprecipitated by incubation for2hat4 °C with the b-galactosidase expression vector pCH110 (Pharmacia-LKB). The effect M2 Flag monoclonal antibody (IBI-Kodak) bound to protein G-Sepha- of co-transfection with 100 ng of expression vectors for p38 MAP kinase, rose (Pharmacia-LKB Biotechnology, Inc). The immunoprecipitates MKK3, MKK4, MKK6, or the empty expression vector was examined. were washed twice with buffer A and twice with kinase buffer (25 mM The cells were harvested 48 h post-transfection. The b-galactosidase HEPES, pH 7.4, 25 mM b-glycerophosphate, 25 mM MgCl , 0.5 mM and luciferase activity in the cell lysates was measured as described dithiothreitol, 0.1 mM sodium orthovanadate). previously (45). Protein kinase assays were performed using recombinant protein kinases and protein kinase immunoprecipitates. The reactions were RESULTS initiated by addition of 1 mg of substrate proteins and 50 mM [g- P]ATP Molecular Cloning of p38b2 MAP Kinase—To identify novel (10 Ci/mmol) in a final volume of 40 ml of kinase buffer. The phospho- members of the human p38 MAP kinase group, we used a rylation reaction was linear with time for at least 40 min. The reactions human p38a MAP kinase cDNA as a probe to screen a human were terminated after 30 min at 30 °C by addition of Laemmli sample buffer. Phosphorylation of the substrate proteins was examined after fetal brain cDNA library. Two cDNA clones related to p38a SDS-polyacrylamide gel electrophoresis (PAGE) by autoradiography MAP kinase were identified. Partial sequence analysis demon- and PhosphorImager analysis. strated that these clones were identical to the p38b MAP ki- Western Blot Analysis—Proteins were fractionated by SDS-PAGE, nase reported by Jiang et al. (23). However, following comple- electrophoretically transferred to an Immobilon-P membrane (Millipore tion of the sequence analysis, it was apparent that the novel Inc.), and probed with the M2 monoclonal antibody to the Flag epitope cDNAs differed from p38b MAP kinase. A deletion of 24 base (IBI-Kodak), a monoclonal antibody to MKK4 (Pharmingen), and an affinity-purified polyclonal antibody to phospho-Ser-383 Elk-1 (New pairs was detected in the sequence of the novel clones compared England Biolabs). Immunocomplexes were detected using chemilumi- with p38b MAP kinase. This gap results in the deletion of an nescence (Lumiglo; Kirkegaard & Perry Laboratories). 8-amino acid insertion present in p38b MAP kinase (23). We Measurement of Reporter Gene Expression—Transfection assays designate the novel sequence as p38b2 and the previously were performed using CHO cells and the LipofectAMINE method (Life reported sequence p38b1 (Fig. 1). Technologies, Inc.). The cells were co-transfected with 0.2 mg of pGAL4- It is possible that the 8-amino acid deletion was the result of Elk-1 (45) and 0.2 mg of the reporter plasmid pG5E1bLuc (49). Trans- fection efficiency was normalized by co-transfection of the cells with the alternative splicing. To test this hypothesis, we performed RT- Molecular Cloning of Human p38b2 MAP Kinase 1743 FIG.3. Substrate specificity of p38 MAP kinases. A, epitope- tagged p38 MAP kinases were transfected in COS cells. The cells were then irradiated with 80 J of UV-C per m (1) or left untreated (2). The p38 MAP kinases were immunopurified and substrate phosphorylation by each p38 MAP kinase was examined in an immuncomplex protein kinase assay. The level of expression of the epitope-tagged p38 MAP FIG.2. The p38b2 protein kinase is a novel MAP kinase. A, kinases was examined by Western blot analysis using the M2 mono- recombinant GST-p38b1 and p38b2 were incubated with [g P]ATP clonal antibody (lower panel). B, the phosphorylation of ATF2 was and buffer (2) or GST-ATF2 (ATF2)(1). The phosphorylation reaction examined using immunopurified p38 MAP kinases prepared from was terminated by addition of Laemmli sample buffer, and the phos- COS-7 cells exposed to UV radiation. The effect of replacement of the phorylated proteins were detected after SDS-PAGE by autoradiogra- phosphorylation sites Thr-69 and Thr-71 with Ala residues is presented. phy. The ATF2, p38b1, and p38b2 are indicated with arrowheads. B, COS-7 cells expressing epitope-tagged p38a, p38b1, p38b2, and p38g The absence of protein kinase activity detected for p38b1 in were exposed to 80 J of UV-C per m (1) and compared with control cells (2). The p38 MAP kinases were isolated by immunoprecipitation and vitro may not represent the activity of this protein kinase in used for protein kinase assays with ATF2 as the substrate (upper vivo. We therefore tested the activity of p38b1 and p38b2 in panel). The phosphorylation reaction was initiated by the addition of vivo. Equal amounts of these p38 MAP kinase isoforms were [g- P]ATP and ATF2. The level of expression of the epitope-tagged p38 expressed in COS-7 cells (Fig. 2B). Exposure to UV-C radiation MAP kinases was examined by Western blot analysis using the M2 monoclonal antibody (lower panel). C, epitope-tagged p38a, p38b1, caused increased protein kinase activity of p38b2, but not p38b2, and p38g were immunoprecipitated from cells co-transfected p38b1 (Fig. 2B). Control experiments demonstrated that UV- with empty vector (2) or activated MKK6 (1) and used for kinase activated p38b2 was less active than UV-activated p38a or assays with ATF2 as a substrate (upper panel). The level of expression p38g (Fig. 2B). of the epitope-tagged MKK6 and the p38 MAP kinases was examined by Western blot analysis using the M2 monoclonal antibody (lower panel). The absence of p38b1 activity and the low level of p38b2 activity compared with p38a and p38g may indicate that UV PCR using primers that span the deletion (59-TCCATCGAG- radiation is a poor activator of p38b1 and p38b2 protein kinase GACTTCAGCGAAGTG-39 and 59-GCCTGGCGCGCCAGCCC- activity. We therefore examined the activity of the p38b MAP GAAATC-39). Sequence analysis of the products of amplifica- kinase isoforms in co-transfection experiments with constitu- tion of human brain mRNA led to the identification of p38b2, tively activated MKK6, a strong activator of p38 MAP kinases but not p38b1. These data demonstrate that in human brain (11). These experiments demonstrated that the activation of p38b2 is the major p38b isoform. It is possible that p38b1 may p38b2 MAP kinase was similar to the activation of p38a and be expressed in other tissues. p38g MAP kinases (Fig. 2C). In contrast, the p38b1 MAP Biochemical Characterization of p38b2 MAP Kinase Activity kinase isoform was inactive in this assay (Fig. 2C). in Vivo and in Vitro—The phosphorylation of ATF2 by p38a Substrate Phosphorylation by p38b2 MAP Kinase—We im- MAP kinase has been studied in detail (11, 12). We therefore munopurified p38a, p38b2, and p38g MAP kinases from control tested whether ATF2 was a substrate for p38b2. In vitro pro- and UV-irradiated cells. The amount of each Flag-tagged p38 tein kinase assays using recombinant p38b2 demonstrated MAP kinase was examined by immunoblot analysis using the that this protein kinase did autophosphorylate (Fig. 2A). In M2 monoclonal antibody. An equal amount of each p38 MAP contrast, control studies using recombinant p38b1 did not dem- kinase isoform was used for in vitro protein kinase assays using onstrate autophosphorylation (Fig. 2A). Addition of ATF2 re- different substrates. This analysis demonstrated that ATF2, sulted in phosphorylation by p38b2, but not by p38b1 (Fig. 2A). Elk-1, and MBP were phosphorylated by p38b2 (Fig. 3A). These data suggest that p38b2 is a more active protein kinase ATF2, Elk-1, and MBP were also phosphorylated by p38a and than p38b1. p38g MAP kinases (Fig. 3A). The extent of substrate phospho- 1744 Molecular Cloning of Human p38b2 MAP Kinase rylation by p38b2 was less than p38a and p38g. However, the fold-activation of p38b2 activity was similar to that detected for p38a and p38g (Fig. 3A). These data indicate that the substrate specificity of these p38 MAP kinase isoforms was similar. To further examine the substrate specificity of p38b2 MAP kinase, we examined the phosphorylation of two other sub- strates for stress-activated MAP kinases. First, the transcrip- tion factor c-Jun, which is phosphorylated by JNK (3). We found that c-Jun was not phosphorylated by any of the p38 MAP kinase isoforms tested (data not shown). In a second series of experiments, we examined the phosphorylation of the protein kinase Mapkap-K2, which is reported to be a substrate for p38 MAP kinase (16, 17). These studies indicated that p38a MAP kinase, but not p38b2orp38g, phosphorylated Map- kap-K2 (Fig. 3A). Thus, the p38b2 MAP kinase substrate spec- ificity differs from p38a MAP kinase. The results of substrate analysis indicate that the substrate specificity of p38b2 MAP kinase was similar to p38g (Fig. 3A). However, this analysis of substrate phosphorylation does not take account of the sites of phosphorylation of each protein. We therefore performed more detailed analysis of p38b2 MAP ki- nase activity using the transcription factor ATF2 as the sub- strate. ATF2 was found to be a substrate for p38a, p38b2, and p38g MAP kinase (Fig. 3A). We have previously reported that FIG.4. The p38b2 MAP kinase is inhibited by SB203580. The effect of SB203580 on p38 MAP kinases was tested in an in vitro protein p38a MAP kinase phosphorylates ATF2 on Thr-69 and Thr-71 kinase assay. Recombinant p38a, p38b2, and p38g MAP kinases were (12). Mutational analysis confirmed this conclusion. Replace- incubated without SB203580 (lanes 1 and 2) or with 0.1, 1, and 10 mM ment of Thr-69 or Thr-71 with Ala caused decreased phospho- of the drug (lanes 3, 4, and 5, respectively) in the presence of [g- P]ATP rylation of ATF2, while replacement of both phosphorylation (lanes 1–5) and ATF2 as a substrate (lanes 2–5). The reaction was sites with Ala eliminated ATF2 phosphorylation by p38a MAP terminated by the addition of Laemmli sample buffer, and the phos- phorylated proteins were detected after SDS-PAGE by autoradiogra- kinase (Fig. 3B). In contrast, experiments using p38g MAP phy. The rate of phosphorylation was quantitated by PhosphorImager kinase demonstrated that the replacement of Thr-71 with Ala analysis and is presented as p38 MAP kinase activity relative to the caused a marked decrease in ATF2 phosphorylation, while kinase activity in the absence of SB203580 (1.0). replacement of Thr-69 with Ala increased ATF2 phosphoryla- tion (Fig. 3B). These data suggest that either Thr-71 is the effects of pyridinyl imidazole derivatives on cultured cells (4, 5). major site of ATF2 phosphorylation by p38g MAP kinase, or The p38b2 MAP kinase is therefore likely to be a physiologi- that the phosphorylation of Thr-71 precedes the phosphoryla- cally relevant mediator of the p38 MAP kinase signal trans- tion of other sites by p38g MAP kinase. Comparative studies duction pathway. using p38b2 MAP kinase demonstrate that this MAP kinase The p38b2 MAP Kinase Is Activated by Proinflammatory isoform differs from both p38a and p38g MAP kinase isoforms. Cytokines and Environmental Stress—The p38a and p38g MAP Replacement of Thr-69 with Ala caused a small decrease in kinases are regulated by numerous extracellular stimuli, in- ATF2 phosphorylation, replacement of Thr-71 with Ala caused cluding proinflammatory cytokines and environmental stress a larger decrease in ATF2 phosphorylation, and replacement of (38). We compared the regulation of p38b2 MAP kinase with both residues caused a marked reduction, but not the elimina- the p38a and p38g MAP kinases. HeLa cells were transfected tion, of ATF2 phosphorylation by p38b2 (Fig. 3B). These data with epitope-tagged p38a, p38b2, and p38g MAP kinases and suggest that both Thr-69 and Thr-71 are phosphorylated by exposed to different extracellular stimuli. The activity of each p38b2 MAP kinase. However, the phosphorylation of ATF2 by p38 MAP kinase isoform was detected by measurement of p38b2 MAP kinase following replacement of both Thr-69 and protein kinase activity in an immune complex kinase assay Thr-71 with Ala residues indicate that p38b2 phosphorylates a using ATF2 as the substrate. The proinflammatory cytokines novel site on ATF2. Together, these data demonstrate that the TNF-a and IL-1a, environmental stress (UV irradiation and substrate specificity of p38b2 MAP kinase differs from the osmotic shock), and treatment with anisomycin (an inhibitor of p38a and p38g MAP kinase isoforms. protein synthesis) caused a marked increase in the activity of Effect of a Pyridinyl Imidazole Drug on p38b2 MAP Kinase p38b2 MAP kinase (Fig. 5). The strongest activation of p38b2 Activity—We examined the effect of the pyridinyl imidazole was caused by the exposure of cells to UV radiation. Although derivative SB203580 (4, 5) on the protein kinase activity of ATF2 phosphorylation by p38b2 was consistently less than p38b2 MAP kinase. This drug has previously been shown to that observed in kinase assays with p38a or p38g, the fold- inhibit p38a MAP kinase activity (4, 5). Here we demonstrate increase in protein kinase activity caused by UV radiation was that SB203580 inhibits p38b2 MAP kinase activity (Fig. 4). similar for each p38 MAP kinase isoform (Fig. 5). These data The dose response of inhibition of protein kinase activity was demonstrate that p38b2, like p38a and p38g, is activated in similar in experiments using p38a and p38b2 MAP kinase (Fig. vivo by a stress-induced signal transduction pathway. 4). In contrast, p38g MAP kinase was not inhibited by Selective Activation of p38 MAP Kinases by MAP Kinase SB203580. The insensitivity of p38g MAP kinase to inhibition Kinases—The p38 MAP kinases are activated in response to by SB203580 observed in this study differs from the results of extracellular stimuli by dual phosphorylation on Thr and Tyr one previous study (26), but is in agreement with more recent by the MAP kinases kinases MKK3 (30), MKK4 (30, 34), and studies by other investigators (25). As the p38a and p38b2 MKK6 (11, 31–33). We therefore tested the effect of MKK3, isoforms demonstrate similar inhibition by SB203580, both of MKK4, and MKK6 on p38b2 MAP kinase activity in co-trans- these MAP kinases could account for the previously reported fection assays in vivo. Control experiments were performed Molecular Cloning of Human p38b2 MAP Kinase 1745 FIG.5. The p38b2 MAP kinase is activated by pro-inflamma- tory cytokines and environmental stress. The activity of epitope- tagged p38a, p38b2 and p38g MAP kinases was measured in immune complex protein kinase assays using [g- P]ATP and ATF2 as substrate. The effect of treatment of Hela cells (30 min) with 10 ng/ml TNF-a,10 ng/ml IL-1a, 300 mM sorbitol, 10 mg/ml anisomycin, and 80 J of UV-C per m was examined. The phosphorylated ATF2 was detected by au- toradiography (15 min). To detect the lower level of p38b2 activity, a longer autoradiographic exposure (45 min) of the phosphorylated ATF2 is also presented. The rate of phosphorylation was quantitated by FIG.6. Selective activation of p38 MAP kinase isoforms by PhosphorImager analysis and is presented as the percentage of p38 MAP kinase kinases in vivo. The ability of MKK3 (panel A), MKK4 MAP kinase activity relative to cells treated with UV radiation (100%). (panel B), and MKK6 (panel C) to activate p38a, p38b2, and p38g was tested in co-transfection assays. COS-7 cells were transfected with epitope-tagged p38a, p38b2, or p38g together with an empty vector using p38a and p38g MAP kinases. MKK3 caused strong acti- (Control) or an expression vector encoding epitope-tagged constitutively vation of p38a, a lower level of activation of p38g, and did not activated MKK3, MKK4, and MKK6. The p38 MAP kinase activity was activate p38b2 (Fig. 6A). Similarly, MKK4 activated p38a measured in an immune complex kinase assay using ATF2 as the substrate. The level of expression of the p38 MAP kinases and the MAP strongly, weakly activated p38g, and did not activate p38b2 kinase kinases was examined by Western blot analysis. (Fig. 6B). In contrast, MKK6 caused strong activation of p38a, p38b2, and p38g MAP kinases (Fig. 6C). The effect of MKK3 cause potent stimulation of co-transfected p38 MAP kinase and MKK4 to activate p38a and p38g, but not p38b2, indicates activity (11). Activated MKK3 can therefore be used as a tool to that the regulation of p38b2 MAP kinase differs from the other test the contribution of specific p38 MAP kinases isoforms on p38 MAP kinases. The selective effect of MAP kinase kinases to cellular responses. Co-transfection assays demonstrated that regulate p38b2 MAP kinase activity suggests that extracellular activated MKK3 did increase Elk-1-dependent luciferase gene stimuli may selectively regulate p38 MAP kinase isoforms. expression when co-transfected with p38 a and p38g MAP ki- The p38b2 MAP Kinase Is a Substrate for MKK6, but Not nases (Fig. 8, A and C). In contrast, p38b2 MAP kinase did not MKK3—The effect of MKK3 to activate p38a and p38g MAP increase MKK3-stimulated Elk-1 transcriptional activity (Fig. kinases, but not p38b2, could be accounted for by many mech- 8B). Control experiments using activated MKK6 demonstrated anisms. One possibility is that p38b2 is not a substrate for that p38 a, p38b2, and p38g MAP kinases increased MKK6- MKK3. To test this hypothesis, we examined the phosphoryla- stimulated Elk-1 transcriptional activity (Fig. 8, A–C). Consist- tion of p38 MAP kinase isoforms by MKK3 and MKK6 (Fig. 7). ent with these data, MKK6 caused a similar level of phospho- In vitro protein kinase assays demonstrated the autophospho- rylation of Elk-1 by each of the three p38 MAP kinases in vitro rylation of MKK3, but not MKK6, as described previously (11, (Fig. 8D) and in vivo (Fig. 8E). Similar data were obtained in 30). MKK6 caused strong phosphorylation of p38a and p38b2 experiments using the transcription factor ATF2 as a p38 MAP MAP kinase, and caused a lower level of phosphorylation of kinase substrate (data not shown). p38g MAP kinase (Fig. 7B). In contrast, MKK3 caused a sim- Together, these data indicate that MKK6 can couple to p38a, ilar level of phosphorylation of p38a and p38g MAP kinases, p38b2, and p38g MAP kinases to initiate a biological response, but no phosphorylation of p38b2 (Fig. 7A). These data demon- while MKK3 can couple only to p38a and p38g MAP kinases. strate that MKK3 and MKK6 differentially phosphorylate p38 These data are consistent with the potent activation of p38b2 MAP kinase isoforms. Furthermore, these data indicate that by MKK6 and the ineffective activation of p38b2 by MKK3 in p38b2 MAP kinase is a substrate for MKK6, but not MKK3. vitro and in vivo (Figs. 6 and 7). Transcriptional Regulation by the MKK6-p38b2 MAP Kinase DISCUSSION Signaling Pathway—The constitutively active form of MKK3 does not activate endogenous p38 MAP kinase in transient The stress-activated MAP kinases include the JNK and p38 transfection assays (11). In contrast, activated MKK3 does groups (3). The JNK group consists of 10 members that are 1746 Molecular Cloning of Human p38b2 MAP Kinase insertion present in p38b1 may be derived by post-transcrip- tional processing of the p38b2 transcript. Further studies are required to resolve this issue. Our studies of the origin of p38b1 have been limited because we have been unable to detect p38b1 by RT-PCR analysis of human mRNA. No difficulty was experienced in the detection of p38b2. We interpret these data to indicate that p38b2isthe major p38b MAP kinase isoform that is expressed in many human tissues. The expression of the p38b1 isoform may be restricted to specific tissues. The p38b2 MAP Kinase Is a Stress-activated Protein Ki- nase—The p38b2 MAP kinase, like the p38a and p38g MAP kinase isoforms, is activated by treatment of cells with proin- flammatory cytokines (e.g. TNF and IL-1) or by exposure of cells to environmental stress (e.g. UV radiation and osmotic shock) (Fig. 5). The p38b2 MAP kinase shares overlapping, but distinct, substrate specificity with the p38a and p38g MAP kinase isoforms (Fig. 2A). The p38b2 MAP kinase was not observed to phosphorylate c-Myc, IkB, Max, Smad1, c-Fos, and NFAT (data not shown). However, p38b2 was found to phos- phorylate the p38a and p38g substrates ATF2, Elk-1, and MBP FIG.7. Selective phosphorylation of p38 MAP kinase isoforms (Fig. 3A). Mutational analysis of the sites of ATF2 phosphoryl- by MAP kinase kinases. The effect of recombinant MKK3 (panel A) ation demonstrated that the substrate specificity of p38b2 dif- and MKK6 (panel B) to phosphorylate p38 MAP kinases was examined fers from p38a and p38g (Fig. 3B). in an in vitro protein kinase assay using recombinant p38a, p38b2, and p38g MAP kinases. The reactions were initiated by addition of Intriguingly, we have not been able to detect any protein [g- P]ATP. The reaction products were examined by SDS-PAGE and kinase activity in experiments using the p38b1 MAP kinase. autoradiography. The p38 MAP kinase isoforms are indicated by aster- Similar negative data were obtained in vitro and in vivo (Fig. isks (*). The MKK3 and MKK6 are indicated by arrowheads. 2). The lack of activity of p38b1 is in contradiction with studies by Jiang et al. (23) who reported that p38b1 is a constitutively derived by alternative splicing of three genes (50). These JNK activated kinase that preferentially phosphorylates ATF2. The isoforms differ in their tissue distribution and in their interac- reason for this discrepancy is unclear, as we did not detect any tion with substrate proteins (50). It has therefore been pro- phosphorylation of ATF2 by recombinant p38b1 in vitro (Fig. posed that individual JNK isoforms may mediate distinct phys- 1A), nor have we been able to activate transfected p38b1byUV iological responses (50). Similarly, the p38 group of stress- irradiation (Fig. 2B) or by cotransfection with activated MKK6 activated MAP kinases consists of multiple isoforms (1). These (Fig. 2C). Further studies are required to resolve this isoforms include p38a (4, 12, 16, 17, 22), p38b (23), and p38g discrepancy. (24 –27). Recent studies indicate the presence of a fourth p38 Northern blot analysis of p38a and p38b MAP kinases dem- MAP kinase isoform, p38d (28, 29). In addition, alternatively onstrates that the tissue distribution of these isoforms is very spliced forms of p38a MAP kinase have been described (4, 51). similar (23). Consequently, the inhibition of both enzymes by The existence of multiple p38 MAP kinase isoforms provides SB203580 (Fig. 4) suggests that some of the physiological func- the potential for the generation of stimulus-specific and cell tions attributed to p38a, based on the effects of pyridinyl im- type-specific responses to activation of the p38 MAP kinase idazole drugs, could be mediated by p38b2 MAP kinase. There- signaling pathway. The identification of p38 MAP kinase iso- fore, p38b2 may contribute to the regulation of the production forms and their mechanism of activation by MAP kinase ki- of TNF and IL-1 by monocytes in response to lipopolysaccha- nases represents one step that is required for understanding ride (4), the induction of the IL-6 gene expression by TNF in the physiological role of p38 MAP kinases in mammalian cells. fibroblasts (6), or any other cellular responses blocked by Here we describe a novel p38 MAP kinase isoform (p38b2) that SB203580 (5). is selectively activated by the MAP kinase kinase MKK6. MKK3 and MKK6 Differentially Activate p38 MAP Kinase The p38b2 Protein Kinase Is a Novel MAP Kinase—We re- Isoforms—The p38 MAP kinases kinases MKK3, MKK4, and port the molecular cloning of p38b2 MAP kinase, a novel hu- MKK6 selectively activate the p38a, p38b2, and p38g MAP man stress-activated protein kinase. This enzyme is most sim- kinase isoforms (Fig. 9). MKK6 activates p38a, p38b2, and ilar to the previously characterized p38b MAP kinase (p38b1) p38g, while MKK3 and MKK4 only activate p38a and p38g (23) and may be derived from the same gene by alternative (Fig. 6). The lack of activation of p38b2 by MKK3 is explained splicing. The p38b2 MAP kinase contains a 24-base pair dele- by its inability to phosphorylate p38b2, while MKK6 phospho- tion within the coding region of p38b1 MAP kinase. This dele- rylates all three isoforms (Fig. 7). The specificity of MKK3 and tion represents a significant difference between these p38b MKK6 to differentially activate these p38 MAP kinase isoforms MAP kinase isoforms. The p38b1 MAP kinase contains an was confirmed by analysis of the effects of MKK3 and MKK6 on 8-amino acid insertion in the kinase domain that is absent in Elk-1-dependent gene expression (Fig. 8). p38b2 MAP kinase (Fig. 1). It is most likely that p38b1 and p38b2 MAP kinases repre- The observation that MAP kinase kinases differentially reg- ulate p38 MAP kinase isoforms has important implications for sent alternatively spliced forms of the same gene. However, sequence analysis of a p38b2 MAP kinase genomic clone dem- the specificity of signal transduction mechanisms. Stimuli that selectively activate MAP kinases kinases would lead to the onstrated that the site of the p38b1 insertion was present within an exon (not at an exon boundary) and that the 24 base activation of different groups of p38 MAP kinase isoforms. This pair p38b1 insertion sequence was not detected in the genomic selective activation of MAP kinases provides a mechanism for clone (data not shown). These data suggest that p38b1 and the generation of stimulus-specific responses of cells to their p38b2 may be encoded by different genes. Alternatively, the environment. However, this mechanism does require that spe- Molecular Cloning of Human p38b2 MAP Kinase 1747 FIG.9. The p38 MAP kinase kinases MKK3, MKK4, and MKK6 cause selective activation of p38 MAP kinase isoforms. The MAP kinase kinase MKK6 phosphorylates and strongly activates p38a, p38b2, and p38g. MKK3 activates p38a and p38g, but not p38b2. The MAP kinase kinase MKK4 activates p38a and does not activate p38b2. MKK4 is also a weak activator p38g. The differential activation of these p38 MAP kinase isoforms may lead to cell type-specific and stimulus- specific cellular responses. cific stimuli cause the differential activation of MAP kinase kinases. It is established that nonrelated MAP kinase kinases are selectively activated in response to the treatment of cells with different stimuli (3). For example, MEK1 is activated by phor- bol ester and MKK3 is activated by UV radiation (30). How- ever, detailed comparative studies of related MAP kinases ki- nases have not yet been completed. It is therefore not clear whether the selective activation of related MAP kinase kinases (e.g. MKK3 and MKK6) is a common event or whether it is unusual. Further studies are required to provide an answer to this question. A precedent for the selective activation of related MAP ki- nases kinases has been established by previous studies of the ERK pathway (52). It has been shown that a proline-rich region in MEK1 and MEK2, the upstream activators of ERK, is nec- essary for recognition and activation by Raf family kinases (53). Phosphorylation of Thr-292 in the proline-rich region of MEK1 regulates the kinetics of inactivation of MEK1 following stim- ulation by growth factors (53). MEK2 lacks this regulatory phosphorylation site and is inactivated more rapidly than MEK1 (53). This regulatory phosphorylation of the proline-rich region of MEK1 provides a mechanism for the differential activation of MEK1 and MEK2 by serum and other stimuli (53). Recent studies have demonstrated the differential regulation of the p38 MAP kinase kinases MKK3 and MKK6. Treatment of Jurkat cells with FAS ligand causes caspase-dependent ac- tivation of the p38 MAP kinase signal transduction pathway (54). FAS-ligation activates MKK6, but not MKK3, in these cells with kinetics which correlated with the onset of FAS- induced apoptosis (55). Constitutively activated MKK6 in- creased the number of apoptotic cells, while dominant-negative MKK6 increased the number of surviving cells following FAS cross-linking (55). These data indicate that FAS ligation selec- tively activates the MKK6, but not MKK3, signaling pathway. These data suggest that MKK3 and MKK6 can be specifically activated by extracellular stimuli and that they have distinct roles. It is likely that such selective activation of p38 MAP kinase kinase isoforms occurs in response to other stimuli and on Elk-1 phosphorylation in vitro. Elk-1 phosphorylation was examined using epitope-tagged p38 MAP kinase isoforms immunoprecipitated from COS cells co-transfected with empty vector (2) or activated MKK6 FIG.8. Regulation of gene expression by p38 MAP kinase iso- (1). The level of expression of epitope-tagged p38 MAP kinases and forms. A–C, effect of p38 MAP kinase isoforms on Elk-1 transcriptional MKK6 was examined by Western blot analysis. E, effect of p38 MAP activity. Elk-1-dependent gene expression was examined in CHO cells kinase isoforms on Elk-1 phosphorylation in vivo. COS cells were co- co-transfected with the b-galactosidase expression vector pCH110, the transfected with Elk-1 and p38 MAP kinase isoforms with empty vector reporter plasmid pG5E1bLuc, and an expression vector for the GAL4 (2) or activated MKK6 (1). The level of expression of epitope-tagged DNA binding domain (residue 1–147) fused to the transcription factor p38 MAP kinases, MKK6, and Elk-1 was examined by Western blot Elk-1 (residue 307– 428). 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Published: Jan 1, 1998

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