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

Hervé Enslen, Joël Raingeaud, and Roger J. DavisDagger

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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of mitogen-activated protein (MAP) kinases. We report the molecular cloning of a novel isoform of p38 MAP kinase, p38beta 2. This p38 MAP kinase, like p38alpha , is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38alpha , p38beta 2, and p38gamma MAP kinase isoforms, while MKK3 activates only p38alpha and p38gamma MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Mitogen-activated protein (MAP)1 kinases are proline-directed protein kinases that mediate the effects of numerous extracellular stimuli on a wide array of biological processes, such as cellular proliferation, differentiation, and death (1). Three groups of mammalian MAP kinases have been studied in detail: the extracellular signal-regulated kinases (ERK) (2), the c-Jun NH2-terminal kinases (JNK) (3), and the p38 MAP kinases (3). The ERKs are robustly activated by growth factors and phorbol ester, but are only weakly activated by cytokines and environmental stress. In contrast, JNK and p38 MAP kinases are strongly activated by cytokines and environmental stress, but are poorly activated by growth factors and phorbol ester.

Recently, progress toward understanding the physiological role of the p38 MAP kinases has been achieved through the use of drugs that bind p38 MAP kinase (4, 5). These drugs are pyridinyl imidazole derivatives that inhibit p38 MAP kinase activity (4, 5). Studies using these drugs indicate that p38 MAP kinase is required for lipopolysaccharide-induced production of IL-1 and TNF in monocytes (4), the induction of IL-6 and granulocyte-macrophage/colony-stimulating factor transcription by TNF (6), the proliferation of T cells in response to IL-2 and IL-7 (7), and the stress-induced transcription of c-jun and c-fos in fibroblasts (8). The targets of p38 MAP kinase that mediate these responses are poorly characterized. However, biochemical studies indicate that p38 MAP kinase signaling pathway activates the transcription factors CREB and ATF1 (9, 10), ATF2 (11, 12), CHOP (13), and MEF-2C (14). The p38 MAP kinase also activates other protein kinases, such as Mapkap-2 (15-17), Mapkap-3 (18, 19), and Mnk1/2 (20, 21).

The p38 MAP kinase group includes the isoforms p38alpha (4, 12, 16, 17, 22), p38beta (23), and p38gamma (24-27). Recent studies indicate the presence of a fourth p38 MAP kinase isoform, p38delta (28, 29). These p38 MAP kinases are widely expressed in many 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 cascade (1). Components of this signaling pathway include the MAP kinase kinases MKK3 (30) and MKK6 (11, 31-33). It is also possible that MKK4 contributes to the activation of p38 MAP kinase. In vitro studies demonstrate that MKK4 activates both JNK and p38 MAP kinases (30, 34). However, the role of MKK4 as an activator of p38 MAP kinase in vivo is unclear (35).

The activation of MKK3 and MKK6 is regulated by phosphorylation on Ser and Thr residues within subdomain VIII by MAP kinase kinase kinases (MKKK) (1). Further studies are required to define the function and specificity of MKKKs that cause activation of the p38 MAP kinase pathway. However, one candidate MKKK for the p38 MAP kinase signaling pathway is TAK1, which has been reported to activate MKK3 and MKK6 (36-38). Other MKKKs, which activate both JNK and p38 MAP kinases, include ASK-1 (39) and the mixed-lineage kinase MLK-3 (40-42). Other MKKKs that activate the JNK signaling pathway, for example MEKK1, do not cause activation of p38 MAP kinase (30, 34).

The expression of multiple p38 MAP kinase isoforms in mammalian tissues suggests that these MAP kinases may differ in their physiological function. These p38 MAP kinases may be coupled to different upstream signaling pathways. This would enable the activation of specific p38 MAP kinase isoforms in response to different stimuli. Alternatively, these p38 MAP kinase isoforms may differ in their substrate specificity. Such differences could allow coupling of different p38 MAP kinase isoforms to different signal transduction targets.

The purpose of this study was to examine the p38beta MAP kinase signal transduction pathway. We find that p38beta MAP kinase (23) is not a functional protein kinase in vitro or in vivo. However, a novel p38beta MAP kinase isoform (p38beta 2) that was isolated from a human brain cDNA library encoded a functional protein kinase. This novel p38 MAP kinase isoform was inhibited by pyridinyl imidazole drugs. The MAP kinase kinase MKK6 activated p38alpha , p38beta 2, and p38gamma , while MKK3 activated only p38alpha and p38gamma . The lack of activation of p38beta 2 by MKK3 was due to its inability to phosphorylate p38beta 2. These data demonstrate that the p38beta 2 MAP kinase is selectively activated by MKK6. We conclude that different p38 MAP kinase isoforms are regulated by overlapping and distinct signal transduction pathways.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- IL-1alpha and TNF-alpha were from Genzyme Corp. MBP was from Sigma. [gamma -32P]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. Recombinant Mapkap-K2 was obtained from Upstate Biotechnology Inc. The drug SB203580 was provided by Dr. M. S.-S. Su, Vertex Pharmaceuticals Inc. Mammalian expression vectors for p38alpha , 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 p38beta 1 (23) was provided by Dr. J. Han (The Scripps Research Institute). The p38gamma cDNA (24, 26, 27) was prepared by RT-PCR amplification using rat skeletal muscle mRNA as the template.

A human p38alpha cDNA was isolated from a fetal brain library by RT-PCR using primers specific for the 5'- and 3'-untranslated regions. This cDNA was subcloned in the vector pCDNA3 (Invitrogen Inc.) and sequenced. The human p38alpha MAP kinase cDNA was labeled with [32P]phosphate by random priming and was used to screen a human fetal brain library cloned in the phage lambda ZAPII (Stratagene). Two clones related to p38beta 1 (designated p38beta 2) were isolated and sequenced.

The mammalian expression vector and the bacterial expression vector for p38alpha , p38beta 1, p38beta 2, and p38gamma MAP kinase were constructed by subcloning the cDNA in the plasmids pCDNA3 (Invitrogen) and pGSTag (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 maintained in Ham's F-12 medium supplemented with 5% fetal bovine serum. HeLa and COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Plasmid DNA (0.1-1.0 µg) was transfected by the LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturers' recommendations. The cells were harvested after 48 h of incubation.

Immunoprecipitation and Protein Kinase Assays-- Cells were solubilized with buffer A (20 mM Tris (pH 7.5), 10% glycerol, 1% Triton X-100, 0.137 M NaCl, 25 mM beta -glycerophosphate, 2 mM EDTA, 0.5 mM dithiothreitol, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). The extracts were centrifuged at 15,000 × g (15 min at 4 °C). Epitope-tagged protein kinases were immunoprecipitated by incubation for 2 h at 4 °C with the M2 Flag monoclonal antibody (IBI-Kodak) bound to protein G-Sepharose (Pharmacia-LKB Biotechnology, Inc). The immunoprecipitates were washed twice with buffer A and twice with kinase buffer (25 mM HEPES, pH 7.4, 25 mM beta -glycerophosphate, 25 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mM sodium orthovanadate).

Protein kinase assays were performed using recombinant protein kinases and protein kinase immunoprecipitates. The reactions were initiated by addition of 1 µg of substrate proteins and 50 µM [gamma -32P]ATP (10 Ci/mmol) in a final volume of 40 µl of kinase buffer. The phosphorylation reaction was linear with time for at least 40 min. The reactions were terminated after 30 min at 30 °C by addition of Laemmli sample buffer. Phosphorylation of the substrate proteins was examined after SDS-polyacrylamide gel electrophoresis (PAGE) by autoradiography and PhosphorImager analysis.

Western Blot Analysis-- Proteins were fractionated by SDS-PAGE, electrophoretically transferred to an Immobilon-P membrane (Millipore Inc.), and probed with the M2 monoclonal antibody to the Flag epitope (IBI-Kodak), a monoclonal antibody to MKK4 (Pharmingen), and an affinity-purified polyclonal antibody to phospho-Ser-383 Elk-1 (New England Biolabs). Immunocomplexes were detected using chemiluminescence (Lumiglo; Kirkegaard & Perry Laboratories).

Measurement of Reporter Gene Expression-- Transfection assays were performed using CHO cells and the LipofectAMINE method (Life Technologies, Inc.). The cells were co-transfected with 0.2 µg of pGAL4-Elk-1 (45) and 0.2 µg of the reporter plasmid pG5E1bLuc (49). Transfection efficiency was normalized by co-transfection of the cells with the beta -galactosidase expression vector pCH110 (Pharmacia-LKB). The effect of co-transfection with 100 ng of expression vectors for p38 MAP kinase, MKK3, MKK4, MKK6, or the empty expression vector was examined. The cells were harvested 48 h post-transfection. The beta -galactosidase and luciferase activity in the cell lysates was measured as described previously (45).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Molecular Cloning of p38beta 2 MAP Kinase-- To identify novel members of the human p38 MAP kinase group, we used a human p38alpha MAP kinase cDNA as a probe to screen a human fetal brain cDNA library. Two cDNA clones related to p38alpha MAP kinase were identified. Partial sequence analysis demonstrated that these clones were identical to the p38beta MAP kinase reported by Jiang et al. (23). However, following completion of the sequence analysis, it was apparent that the novel cDNAs differed from p38beta MAP kinase. A deletion of 24 base pairs was detected in the sequence of the novel clones compared with p38beta MAP kinase. This gap results in the deletion of an 8-amino acid insertion present in p38beta MAP kinase (23). We designate the novel sequence as p38beta 2 and the previously reported sequence p38beta 1 (Fig. 1).


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Fig. 1.   Comparison of the primary sequence of p38beta 2 MAP kinase with the p38alpha , p38beta 1, and p38gamma MAP kinase isoforms. The primary sequence of human p38beta 2 MAP kinase was deduced from the sequence of cDNA clones. The sequence of p38beta 2 is aligned to the p38alpha , p38beta 1, and p38gamma MAP kinase isoforms. Residues that are identical to p38alpha MAP kinase are indicated with a period (.). The sites of activating phosphorylation (Thr and Tyr) are indicated with asterisks. The cDNA sequence of the human p38beta 2 MAP kinase has been deposited in GenBankTM with accession no. AF031135. The sequences of the p38alpha (4, 12, 16, 17, 22), p38beta (23), and p38gamma (24-27) MAP kinase isoforms have been reported previously.

It is possible that the 8-amino acid deletion was the result of alternative splicing. To test this hypothesis, we performed RT-PCR using primers that span the deletion (5'-TCCATCGAGGACTTCAGCGAAGTG-3' and 5'-GCCTGGCGCGCCAGCCCGAAATC-3'). Sequence analysis of the products of amplification of human brain mRNA led to the identification of p38beta 2, but not p38beta 1. These data demonstrate that in human brain p38beta 2 is the major p38beta isoform. It is possible that p38beta 1 may be expressed in other tissues.

Biochemical Characterization of p38beta 2 MAP Kinase Activity in Vivo and in Vitro-- The phosphorylation of ATF2 by p38alpha MAP kinase has been studied in detail (11, 12). We therefore tested whether ATF2 was a substrate for p38beta 2. In vitro protein kinase assays using recombinant p38beta 2 demonstrated that this protein kinase did autophosphorylate (Fig. 2A). In contrast, control studies using recombinant p38beta 1 did not demonstrate autophosphorylation (Fig. 2A). Addition of ATF2 resulted in phosphorylation by p38beta 2, but not by p38beta 1 (Fig. 2A). These data suggest that p38beta 2 is a more active protein kinase than p38beta 1.


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Fig. 2.   The p38beta 2 protein kinase is a novel MAP kinase. A, recombinant GST-p38beta 1 and p38beta 2 were incubated with [gamma -32P]ATP and buffer (-) or GST-ATF2 (ATF2) (+). The phosphorylation reaction was terminated by addition of Laemmli sample buffer, and the phosphorylated proteins were detected after SDS-PAGE by autoradiography. The ATF2, p38beta 1, and p38beta 2 are indicated with arrowheads. B, COS-7 cells expressing epitope-tagged p38alpha , p38beta 1, p38beta 2, and p38gamma were exposed to 80 J of UV-C per m2 (+) and compared with control cells (-). The p38 MAP kinases were isolated by immunoprecipitation and used for protein kinase assays with ATF2 as the substrate (upper panel). The phosphorylation reaction was initiated by the addition of [gamma -32P]ATP and ATF2. The level of expression of the epitope-tagged p38 MAP kinases was examined by Western blot analysis using the M2 monoclonal antibody (lower panel). C, epitope-tagged p38alpha , p38beta 1, p38beta 2, and p38gamma were immunoprecipitated from cells co-transfected with empty vector (-) or activated MKK6 (+) and used for kinase assays with ATF2 as a substrate (upper panel). The level of expression 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 protein kinase activity detected for p38beta 1 in vitro may not represent the activity of this protein kinase in vivo. We therefore tested the activity of p38beta 1 and p38beta 2 in vivo. Equal amounts of these p38 MAP kinase isoforms were expressed in COS-7 cells (Fig. 2B). Exposure to UV-C radiation caused increased protein kinase activity of p38beta 2, but not p38beta 1 (Fig. 2B). Control experiments demonstrated that UV-activated p38beta 2 was less active than UV-activated p38alpha or p38gamma (Fig. 2B).

The absence of p38beta 1 activity and the low level of p38beta 2 activity compared with p38alpha and p38gamma may indicate that UV radiation is a poor activator of p38beta 1 and p38beta 2 protein kinase activity. We therefore examined the activity of the p38beta MAP kinase isoforms in co-transfection experiments with constitutively activated MKK6, a strong activator of p38 MAP kinases (11). These experiments demonstrated that the activation of p38beta 2 MAP kinase was similar to the activation of p38alpha and p38gamma MAP kinases (Fig. 2C). In contrast, the p38beta 1 MAP kinase isoform was inactive in this assay (Fig. 2C).

Substrate Phosphorylation by p38beta 2 MAP Kinase-- We immunopurified p38alpha , p38beta 2, and p38gamma MAP kinases from control and UV-irradiated cells. The amount of each Flag-tagged p38 MAP kinase was examined by immunoblot analysis using the M2 monoclonal antibody. An equal amount of each p38 MAP kinase isoform was used for in vitro protein kinase assays using different substrates. This analysis demonstrated that ATF2, Elk-1, and MBP were phosphorylated by p38beta 2 (Fig. 3A). ATF2, Elk-1, and MBP were also phosphorylated by p38alpha and p38gamma MAP kinases (Fig. 3A). The extent of substrate phosphorylation by p38beta 2 was less than p38alpha and p38gamma . However, the fold-activation of p38beta 2 activity was similar to that detected for p38alpha and p38gamma (Fig. 3A). These data indicate that the substrate specificity of these p38 MAP kinase isoforms was similar.


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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 m2 (+) or left untreated (-). 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 kinases was examined by Western blot analysis using the M2 monoclonal antibody (lower panel). B, the phosphorylation of ATF2 was examined using immunopurified p38 MAP kinases prepared from COS-7 cells exposed to UV radiation. The effect of replacement of the phosphorylation sites Thr-69 and Thr-71 with Ala residues is presented.

To further examine the substrate specificity of p38beta 2 MAP kinase, we examined the phosphorylation of two other substrates for stress-activated MAP kinases. First, the transcription 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 p38alpha MAP kinase, but not p38beta 2 or p38gamma , phosphorylated Mapkap-K2 (Fig. 3A). Thus, the p38beta 2 MAP kinase substrate specificity differs from p38alpha MAP kinase.

The results of substrate analysis indicate that the substrate specificity of p38beta 2 MAP kinase was similar to p38gamma (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 p38beta 2 MAP kinase activity using the transcription factor ATF2 as the substrate. ATF2 was found to be a substrate for p38alpha , p38beta 2, and p38gamma MAP kinase (Fig. 3A). We have previously reported that p38alpha MAP kinase phosphorylates ATF2 on Thr-69 and Thr-71 (12). Mutational analysis confirmed this conclusion. Replacement of Thr-69 or Thr-71 with Ala caused decreased phosphorylation of ATF2, while replacement of both phosphorylation sites with Ala eliminated ATF2 phosphorylation by p38alpha MAP kinase (Fig. 3B). In contrast, experiments using p38gamma MAP kinase demonstrated that the replacement of Thr-71 with Ala caused a marked decrease in ATF2 phosphorylation, while replacement of Thr-69 with Ala increased ATF2 phosphorylation (Fig. 3B). These data suggest that either Thr-71 is the major site of ATF2 phosphorylation by p38gamma MAP kinase, or that the phosphorylation of Thr-71 precedes the phosphorylation of other sites by p38gamma MAP kinase. Comparative studies using p38beta 2 MAP kinase demonstrate that this MAP kinase isoform differs from both p38alpha and p38gamma MAP kinase isoforms. Replacement of Thr-69 with Ala caused a small decrease in ATF2 phosphorylation, replacement of Thr-71 with Ala caused a larger decrease in ATF2 phosphorylation, and replacement of both residues caused a marked reduction, but not the elimination, of ATF2 phosphorylation by p38beta 2 (Fig. 3B). These data suggest that both Thr-69 and Thr-71 are phosphorylated by p38beta 2 MAP kinase. However, the phosphorylation of ATF2 by p38beta 2 MAP kinase following replacement of both Thr-69 and Thr-71 with Ala residues indicate that p38beta 2 phosphorylates a novel site on ATF2. Together, these data demonstrate that the substrate specificity of p38beta 2 MAP kinase differs from the p38alpha and p38gamma MAP kinase isoforms.

Effect of a Pyridinyl Imidazole Drug on p38beta 2 MAP Kinase Activity-- We examined the effect of the pyridinyl imidazole derivative SB203580 (4, 5) on the protein kinase activity of p38beta 2 MAP kinase. This drug has previously been shown to inhibit p38alpha MAP kinase activity (4, 5). Here we demonstrate that SB203580 inhibits p38beta 2 MAP kinase activity (Fig. 4). The dose response of inhibition of protein kinase activity was similar in experiments using p38alpha and p38beta 2 MAP kinase (Fig. 4). In contrast, p38gamma MAP kinase was not inhibited by SB203580. The insensitivity of p38gamma MAP kinase to inhibition by SB203580 observed in this study differs from the results of one previous study (26), but is in agreement with more recent studies by other investigators (25). As the p38alpha and p38beta 2 isoforms demonstrate similar inhibition by SB203580, both of these MAP kinases could account for the previously reported effects of pyridinyl imidazole derivatives on cultured cells (4, 5). The p38beta 2 MAP kinase is therefore likely to be a physiologically relevant mediator of the p38 MAP kinase signal transduction pathway.


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Fig. 4.   The p38beta 2 MAP kinase is inhibited by SB203580. The effect of SB203580 on p38 MAP kinases was tested in an in vitro protein kinase assay. Recombinant p38alpha , p38beta 2, and p38gamma MAP kinases were incubated without SB203580 (lanes 1 and 2) or with 0.1, 1, and 10 µM of the drug (lanes 3, 4, and 5, respectively) in the presence of [gamma -32P]ATP (lanes 1-5) and ATF2 as a substrate (lanes 2-5). The reaction was terminated by the addition of Laemmli sample buffer, and the phosphorylated proteins were detected after SDS-PAGE by autoradiography. The rate of phosphorylation was quantitated by PhosphorImager analysis and is presented as p38 MAP kinase activity relative to the kinase activity in the absence of SB203580 (1.0).

The p38beta 2 MAP Kinase Is Activated by Proinflammatory Cytokines and Environmental Stress-- The p38alpha and p38gamma MAP kinases are regulated by numerous extracellular stimuli, including proinflammatory cytokines and environmental stress (38). We compared the regulation of p38beta 2 MAP kinase with the p38alpha and p38gamma MAP kinases. HeLa cells were transfected with epitope-tagged p38alpha , p38beta 2, and p38gamma MAP kinases and exposed to different extracellular stimuli. The activity of each p38 MAP kinase isoform was detected by measurement of protein kinase activity in an immune complex kinase assay using ATF2 as the substrate. The proinflammatory cytokines TNF-alpha and IL-1alpha , environmental stress (UV irradiation and osmotic shock), and treatment with anisomycin (an inhibitor of protein synthesis) caused a marked increase in the activity of p38beta 2 MAP kinase (Fig. 5). The strongest activation of p38beta 2 was caused by the exposure of cells to UV radiation. Although ATF2 phosphorylation by p38beta 2 was consistently less than that observed in kinase assays with p38alpha or p38gamma , the fold-increase in protein kinase activity caused by UV radiation was similar for each p38 MAP kinase isoform (Fig. 5). These data demonstrate that p38beta 2, like p38alpha and p38gamma , is activated in vivo by a stress-induced signal transduction pathway.


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Fig. 5.   The p38beta 2 MAP kinase is activated by pro-inflammatory cytokines and environmental stress. The activity of epitope-tagged p38alpha , p38beta 2 and p38gamma MAP kinases was measured in immune complex protein kinase assays using [gamma -32P]ATP and ATF2 as substrate. The effect of treatment of Hela cells (30 min) with 10 ng/ml TNF-alpha , 10 ng/ml IL-1alpha , 300 mM sorbitol, 10 µg/ml anisomycin, and 80 J of UV-C per m2 was examined. The phosphorylated ATF2 was detected by autoradiography (15 min). To detect the lower level of p38beta 2 activity, a longer autoradiographic exposure (45 min) of the phosphorylated ATF2 is also presented. The rate of phosphorylation was quantitated by PhosphorImager analysis and is presented as the percentage of p38 MAP kinase activity relative to cells treated with UV radiation (100%).

Selective Activation of p38 MAP Kinases by MAP Kinase Kinases-- The p38 MAP kinases are activated in response to extracellular stimuli by dual phosphorylation on Thr and Tyr by the MAP kinases kinases MKK3 (30), MKK4 (30, 34), and MKK6 (11, 31-33). We therefore tested the effect of MKK3, MKK4, and MKK6 on p38beta 2 MAP kinase activity in co-transfection assays in vivo. Control experiments were performed using p38alpha and p38gamma MAP kinases. MKK3 caused strong activation of p38alpha , a lower level of activation of p38gamma , and did not activate p38beta 2 (Fig. 6A). Similarly, MKK4 activated p38alpha strongly, weakly activated p38gamma , and did not activate p38beta 2 (Fig. 6B). In contrast, MKK6 caused strong activation of p38alpha , p38beta 2, and p38gamma MAP kinases (Fig. 6C). The effect of MKK3 and MKK4 to activate p38alpha and p38gamma , but not p38beta 2, indicates that the regulation of p38beta 2 MAP kinase differs from the other p38 MAP kinases. The selective effect of MAP kinase kinases to regulate p38beta 2 MAP kinase activity suggests that extracellular stimuli may selectively regulate p38 MAP kinase isoforms.


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Fig. 6.   Selective activation of p38 MAP kinase isoforms by MAP kinase kinases in vivo. The ability of MKK3 (panel A), MKK4 (panel B), and MKK6 (panel C) to activate p38alpha , p38beta 2, and p38gamma was tested in co-transfection assays. COS-7 cells were transfected with epitope-tagged p38alpha , p38beta 2, or p38gamma together with an empty vector (Control) or an expression vector encoding epitope-tagged constitutively activated MKK3, MKK4, and MKK6. The p38 MAP kinase activity was measured in an immune complex kinase assay using ATF2 as the substrate. The level of expression of the p38 MAP kinases and the MAP kinase kinases was examined by Western blot analysis.

The p38beta 2 MAP Kinase Is a Substrate for MKK6, but Not MKK3-- The effect of MKK3 to activate p38alpha and p38gamma MAP kinases, but not p38beta 2, could be accounted for by many mechanisms. One possibility is that p38beta 2 is not a substrate for MKK3. To test this hypothesis, we examined the phosphorylation of p38 MAP kinase isoforms by MKK3 and MKK6 (Fig. 7). In vitro protein kinase assays demonstrated the autophosphorylation of MKK3, but not MKK6, as described previously (11, 30). MKK6 caused strong phosphorylation of p38alpha and p38beta 2 MAP kinase, and caused a lower level of phosphorylation of p38gamma MAP kinase (Fig. 7B). In contrast, MKK3 caused a similar level of phosphorylation of p38alpha and p38gamma MAP kinases, but no phosphorylation of p38beta 2 (Fig. 7A). These data demonstrate that MKK3 and MKK6 differentially phosphorylate p38 MAP kinase isoforms. Furthermore, these data indicate that p38beta 2 MAP kinase is a substrate for MKK6, but not MKK3.


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Fig. 7.   Selective phosphorylation of p38 MAP kinase isoforms by MAP kinase kinases. The effect of recombinant MKK3 (panel A) and MKK6 (panel B) to phosphorylate p38 MAP kinases was examined in an in vitro protein kinase assay using recombinant p38alpha , p38beta 2, and p38gamma MAP kinases. The reactions were initiated by addition of [gamma -32P]ATP. The reaction products were examined by SDS-PAGE and autoradiography. The p38 MAP kinase isoforms are indicated by asterisks (*). The MKK3 and MKK6 are indicated by arrowheads.

Transcriptional Regulation by the MKK6-p38beta 2 MAP Kinase Signaling Pathway-- The constitutively active form of MKK3 does not activate endogenous p38 MAP kinase in transient transfection assays (11). In contrast, activated MKK3 does cause potent stimulation of co-transfected p38 MAP kinase activity (11). Activated MKK3 can therefore be used as a tool to test the contribution of specific p38 MAP kinases isoforms on cellular responses. Co-transfection assays demonstrated that activated MKK3 did increase Elk-1-dependent luciferase gene expression when co-transfected with p38alpha and p38gamma MAP kinases (Fig. 8, A and C). In contrast, p38beta 2 MAP kinase did not increase MKK3-stimulated Elk-1 transcriptional activity (Fig. 8B). Control experiments using activated MKK6 demonstrated that p38alpha , p38beta 2, and p38gamma MAP kinases increased MKK6-stimulated Elk-1 transcriptional activity (Fig. 8, A-C). Consistent with these data, MKK6 caused a similar level of phosphorylation of Elk-1 by each of the three p38 MAP kinases in vitro (Fig. 8D) and in vivo (Fig. 8E). Similar data were obtained in experiments using the transcription factor ATF2 as a p38 MAP kinase substrate (data not shown).


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Fig. 8.  

Regulation of gene expression by p38 MAP kinase isoforms. A-C, effect of p38 MAP kinase isoforms on Elk-1 transcriptional activity. Elk-1-dependent gene expression was examined in CHO cells co-transfected with the beta -galactosidase expression vector pCH110, the reporter plasmid pG5E1bLuc, and an expression vector for the GAL4 DNA binding domain (residue 1-147) fused to the transcription factor Elk-1 (residue 307-428). Transfection efficiency was monitored by measurement of beta -galactosidase expression. The relative luciferase activity detected following co-transfection of the empty vector, activated MKK3, or activated MKK6 together with p38alpha (panel A), p38beta 2 (panel B), or p38gamma (panel C) is presented. D, effect of p38 MAP kinase isoforms 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 (-) or activated MKK6 (+). The level of expression of epitope-tagged p38 MAP kinases and MKK6 was examined by Western blot analysis. E, effect of p38 MAP kinase isoforms on Elk-1 phosphorylation in vivo. COS cells were co-transfected with Elk-1 and p38 MAP kinase isoforms with empty vector (-) or activated MKK6 (+). The level of expression of epitope-tagged p38 MAP kinases, MKK6, and Elk-1 was examined by Western blot analysis. The phosphorylation of Elk-1 was examined by Western blots probed with an antibody that binds Elk-1 phosphorylated at Ser-383. Previous studies have established that the phosphorylation of Elk-1 on Ser-383 contributes to increased transcriptional activity caused by p38 MAP kinase (56, 57).

Together, these data indicate that MKK6 can couple to p38alpha , p38beta 2, and p38gamma MAP kinases to initiate a biological response, while MKK3 can couple only to p38alpha and p38gamma MAP kinases. These data are consistent with the potent activation of p38beta 2 by MKK6 and the ineffective activation of p38beta 2 by MKK3 in vitro and in vivo (Figs. 6 and 7).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The stress-activated MAP kinases include the JNK and p38 groups (3). The JNK group consists of 10 members that are derived by alternative splicing of three genes (50). These JNK isoforms differ in their tissue distribution and in their interaction with substrate proteins (50). It has therefore been proposed that individual JNK isoforms may mediate distinct physiological responses (50). Similarly, the p38 group of stress-activated MAP kinases consists of multiple isoforms (1). These isoforms include p38alpha (4, 12, 16, 17, 22), p38beta (23), and p38gamma (24-27). Recent studies indicate the presence of a fourth p38 MAP kinase isoform, p38delta (28, 29). In addition, alternatively spliced forms of p38alpha MAP kinase have been described (4, 51). The existence of multiple p38 MAP kinase isoforms provides the potential for the generation of stimulus-specific and cell type-specific responses to activation of the p38 MAP kinase signaling pathway. The identification of p38 MAP kinase isoforms and their mechanism of activation by MAP kinase kinases represents one step that is required for understanding the physiological role of p38 MAP kinases in mammalian cells. Here we describe a novel p38 MAP kinase isoform (p38beta 2) that is selectively activated by the MAP kinase kinase MKK6.

The p38beta 2 Protein Kinase Is a Novel MAP Kinase-- We report the molecular cloning of p38beta 2 MAP kinase, a novel human stress-activated protein kinase. This enzyme is most similar to the previously characterized p38beta MAP kinase (p38beta 1) (23) and may be derived from the same gene by alternative splicing. The p38beta 2 MAP kinase contains a 24-base pair deletion within the coding region of p38beta 1 MAP kinase. This deletion represents a significant difference between these p38beta MAP kinase isoforms. The p38beta 1 MAP kinase contains an 8-amino acid insertion in the kinase domain that is absent in p38beta 2 MAP kinase (Fig. 1).

It is most likely that p38beta 1 and p38beta 2 MAP kinases represent alternatively spliced forms of the same gene. However, sequence analysis of a p38beta 2 MAP kinase genomic clone demonstrated that the site of the p38beta 1 insertion was present within an exon (not at an exon boundary) and that the 24 base pair p38beta 1 insertion sequence was not detected in the genomic clone (data not shown). These data suggest that p38beta 1 and p38beta 2 may be encoded by different genes. Alternatively, the insertion present in p38beta 1 may be derived by post-transcriptional processing of the p38beta 2 transcript. Further studies are required to resolve this issue.

Our studies of the origin of p38beta 1 have been limited because we have been unable to detect p38beta 1 by RT-PCR analysis of human mRNA. No difficulty was experienced in the detection of p38beta 2. We interpret these data to indicate that p38beta 2 is the major p38beta MAP kinase isoform that is expressed in many human tissues. The expression of the p38beta 1 isoform may be restricted to specific tissues.

The p38beta 2 MAP Kinase Is a Stress-activated Protein Kinase-- The p38beta 2 MAP kinase, like the p38alpha and p38gamma MAP kinase isoforms, is activated by treatment of cells with proinflammatory 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 p38beta 2 MAP kinase shares overlapping, but distinct, substrate specificity with the p38alpha and p38gamma MAP kinase isoforms (Fig. 2A). The p38beta 2 MAP kinase was not observed to phosphorylate c-Myc, Ikappa B, Max, Smad1, c-Fos, and NFAT (data not shown). However, p38beta 2 was found to phosphorylate the p38alpha and p38gamma substrates ATF2, Elk-1, and MBP (Fig. 3A). Mutational analysis of the sites of ATF2 phosphorylation demonstrated that the substrate specificity of p38beta 2 differs from p38alpha and p38gamma (Fig. 3B).

Intriguingly, we have not been able to detect any protein kinase activity in experiments using the p38beta 1 MAP kinase. Similar negative data were obtained in vitro and in vivo (Fig. 2). The lack of activity of p38beta 1 is in contradiction with studies by Jiang et al. (23) who reported that p38beta 1 is a constitutively activated kinase that preferentially phosphorylates ATF2. The reason for this discrepancy is unclear, as we did not detect any phosphorylation of ATF2 by recombinant p38beta 1 in vitro (Fig. 1A), nor have we been able to activate transfected p38beta 1 by UV irradiation (Fig. 2B) or by cotransfection with activated MKK6 (Fig. 2C). Further studies are required to resolve this discrepancy.

Northern blot analysis of p38alpha and p38beta MAP kinases demonstrates that the tissue distribution of these isoforms is very similar (23). Consequently, the inhibition of both enzymes by SB203580 (Fig. 4) suggests that some of the physiological functions attributed to p38alpha , based on the effects of pyridinyl imidazole drugs, could be mediated by p38beta 2 MAP kinase. Therefore, p38beta 2 may contribute to the regulation of the production of TNF and IL-1 by monocytes in response to lipopolysaccharide (4), the induction of the IL-6 gene expression by TNF in fibroblasts (6), or any other cellular responses blocked by SB203580 (5).

MKK3 and MKK6 Differentially Activate p38 MAP Kinase Isoforms-- The p38 MAP kinases kinases MKK3, MKK4, and MKK6 selectively activate the p38alpha , p38beta 2, and p38gamma MAP kinase isoforms (Fig. 9). MKK6 activates p38alpha , p38beta 2, and p38gamma , while MKK3 and MKK4 only activate p38alpha and p38gamma (Fig. 6). The lack of activation of p38beta 2 by MKK3 is explained by its inability to phosphorylate p38beta 2, while MKK6 phosphorylates all three isoforms (Fig. 7). The specificity of MKK3 and MKK6 to differentially activate these p38 MAP kinase isoforms was confirmed by analysis of the effects of MKK3 and MKK6 on Elk-1-dependent gene expression (Fig. 8).


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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 p38alpha , p38beta 2, and p38gamma . MKK3 activates p38alpha and p38gamma , but not p38beta 2. The MAP kinase kinase MKK4 activates p38alpha and does not activate p38beta 2. MKK4 is also a weak activator p38gamma . The differential activation of these p38 MAP kinase isoforms may lead to cell type-specific and stimulus-specific cellular responses.

The observation that MAP kinase kinases differentially regulate p38 MAP kinase isoforms has important implications for the specificity of signal transduction mechanisms. Stimuli that selectively activate MAP kinases kinases would lead to the activation of different groups of p38 MAP kinase isoforms. This selective activation of MAP kinases provides a mechanism for the generation of stimulus-specific responses of cells to their environment. However, this mechanism does require that specific 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 phorbol ester and MKK3 is activated by UV radiation (30). However, detailed comparative studies of related MAP kinases kinases 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 kinases 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 necessary 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 stimulation 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 activation 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 increased 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 selectively 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 in other cell types.

Conclusions-- The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of MAP kinases. The activation of specific p38 MAP kinase isoforms may lead to cell type-specific and stimulus-specific cellular responses. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38alpha , p38beta 2, and p38gamma MAP kinases, while MKK3 targets p38alpha and p38gamma MAP kinases. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinase isoforms.

    ACKNOWLEDGEMENTS

We thank Dr. J. Han for the p38beta 1 MAP kinase cDNA and for discussions concerning p38beta 1 protein kinase activity; Drs. A. J. Whitmarsh and A. Sharrocks for critical discussions and for providing the plasmid pCMV-Flag-Elk-1; Dr. M. S.-S. Su for the drug SB203580; T. Barrett, J. Cavanagh, and I.-H. Wu for technical assistance; and K. Gemme for administrative assistance.

    FOOTNOTES

* These studies were supported in part by National Institutes of Health, NCI, Grants CA65861 and CA72009.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF031135.

Dagger Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St., Worcester, MA 01605.

1 The abbreviations used are: MAP, mitogen-activated protein; MKK, MAP kinase kinase; MKKK, MAP kinase kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; IL, interleukin; TNF, tumor necrosis factor; GST, glutathione S-transferase; RT, reverse transcriptase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; MBP, myelin basic protein.

    REFERENCES
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