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

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, p38β2. This p38 MAP kinase, like p38α, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38α, p38β2, and p38γ MAP kinase isoforms, while MKK3 activates only p38α and p38γ MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.

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, p38␤2. This p38 MAP kinase, like p38␣, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38␣, p38␤2, and p38␥ MAP kinase isoforms, while MKK3 activates only p38␣ and p38␥ MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.
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 NH 2 -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.
The p38 MAP kinase group includes the isoforms p38␣ (4,12,16,17,22), p38␤ (23), and p38␥ (24 -27). Recent studies indicate the presence of a fourth p38 MAP kinase isoform, p38␦ (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)(32)(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 p38␤ MAP kinase signal transduction pathway. We find that p38␤ MAP kinase (23) is not a functional protein kinase in vitro or in vivo. However, a novel p38␤ MAP kinase isoform (p38␤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 p38␣, p38␤2, and p38␥, while MKK3 activated only p38␣ and p38␥. The lack of activation of p38␤2 by MKK3 was due to its inability to phosphorylate p38␤2. These data demonstrate that the p38␤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.
A human p38␣ cDNA was isolated from a fetal brain library by RT-PCR using primers specific for the 5Ј-and 3Ј-untranslated regions. This cDNA was subcloned in the vector pCDNA3 (Invitrogen Inc.) and sequenced. The human p38␣ MAP kinase cDNA was labeled with [ 32 P]phosphate by random priming and was used to screen a human fetal brain library cloned in the phage ZAPII (Stratagene). Two clones related to p38␤1 (designated p38␤2) were isolated and sequenced.
The mammalian expression vector and the bacterial expression vector for p38␣, p38␤1, p38␤2, and p38␥ 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 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 g) was transfected by the Lipo-fectAMINE reagent (Life Technologies, Inc.) according to the manufacturers' recommendations. The cells were harvested after 48 h of incubation.
Protein kinase assays were performed using recombinant protein kinases and protein kinase immunoprecipitates. The reactions were initiated by addition of 1 g of substrate proteins and 50 M [␥-32 P]ATP (10 Ci/mmol) in a final volume of 40 l 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.
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 g of pGAL4-Elk-1 (45) and 0.2 g of the reporter plasmid pG5E1bLuc (49). Transfection efficiency was normalized by co-transfection of the cells with the ␤-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 ␤-galactosidase and luciferase activity in the cell lysates was measured as described previously (45).

Molecular
Cloning of p38␤2 MAP Kinase-To identify novel members of the human p38 MAP kinase group, we used a human p38␣ MAP kinase cDNA as a probe to screen a human fetal brain cDNA library. Two cDNA clones related to p38␣ MAP kinase were identified. Partial sequence analysis demonstrated that these clones were identical to the p38␤ MAP kinase reported by Jiang et al. (23). However, following completion of the sequence analysis, it was apparent that the novel cDNAs differed from p38␤ MAP kinase. A deletion of 24 base pairs was detected in the sequence of the novel clones compared with p38␤ MAP kinase. This gap results in the deletion of an 8-amino acid insertion present in p38␤ MAP kinase (23). We designate the novel sequence as p38␤2 and the previously reported sequence p38␤1 (Fig. 1).
It is possible that the 8-amino acid deletion was the result of alternative splicing. To test this hypothesis, we performed RT-FIG. 1. Comparison of the primary sequence of p38␤2 MAP kinase with the p38␣, p38␤1, and p38␥ MAP kinase isoforms. The primary sequence of human p38␤2 MAP kinase was deduced from the sequence of cDNA clones. The sequence of p38␤2 is aligned to the p38␣, p38␤1, and p38␥ MAP kinase isoforms. Residues that are identical to p38␣ 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 p38␤2 MAP kinase has been deposited in Gen-Bank TM with accession no. AF031135. The sequences of the p38␣ (4,12,16,17,22), p38␤ (23), and p38␥ (24 -27) MAP kinase isoforms have been reported previously. PCR using primers that span the deletion (5Ј-TCCATCGAG-GACTTCAGCGAAGTG-3Ј and 5Ј-GCCTGGCGCGCCAGCCC-GAAATC-3Ј). Sequence analysis of the products of amplification of human brain mRNA led to the identification of p38␤2, but not p38␤1. These data demonstrate that in human brain p38␤2 is the major p38␤ isoform. It is possible that p38␤1 may be expressed in other tissues.
Biochemical Characterization of p38␤2 MAP Kinase Activity in Vivo and in Vitro-The phosphorylation of ATF2 by p38␣ MAP kinase has been studied in detail (11,12). We therefore tested whether ATF2 was a substrate for p38␤2. In vitro protein kinase assays using recombinant p38␤2 demonstrated that this protein kinase did autophosphorylate ( Fig. 2A). In contrast, control studies using recombinant p38␤1 did not demonstrate autophosphorylation ( Fig. 2A). Addition of ATF2 resulted in phosphorylation by p38␤2, but not by p38␤1 ( Fig. 2A). These data suggest that p38␤2 is a more active protein kinase than p38␤1.
The absence of protein kinase activity detected for p38␤1 in vitro may not represent the activity of this protein kinase in vivo. We therefore tested the activity of p38␤1 and p38␤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 p38␤2, but not p38␤1 (Fig. 2B). Control experiments demonstrated that UVactivated p38␤2 was less active than UV-activated p38␣ or p38␥ (Fig. 2B).
The absence of p38␤1 activity and the low level of p38␤2 activity compared with p38␣ and p38␥ may indicate that UV radiation is a poor activator of p38␤1 and p38␤2 protein kinase activity. We therefore examined the activity of the p38␤ 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 p38␤2 MAP kinase was similar to the activation of p38␣ and p38␥ MAP kinases (Fig. 2C). In contrast, the p38␤1 MAP kinase isoform was inactive in this assay (Fig. 2C).
Substrate Phosphorylation by p38␤2 MAP Kinase-We immunopurified p38␣, p38␤2, and p38␥ 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 p38␤2 (Fig. 3A). ATF2, Elk-1, and MBP were also phosphorylated by p38␣ and p38␥ MAP kinases (Fig. 3A). The extent of substrate phospho-FIG. 2. The p38␤2 protein kinase is a novel MAP kinase. A, recombinant GST-p38␤1 and p38␤2 were incubated with [␥ -32 P]ATP and buffer (Ϫ) or GST-ATF2 (ATF2) (ϩ). The phosphorylation reaction was terminated by addition of Laemmli sample buffer, and the phosphorylated proteins were detected after SDS-PAGE by autoradiography. The ATF2, p38␤1, and p38␤2 are indicated with arrowheads. B, COS-7 cells expressing epitope-tagged p38␣, p38␤1, p38␤2, and p38␥ were exposed to 80 J of UV-C per m 2 (ϩ) and compared with control cells (Ϫ). 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 [␥-32 P]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 p38␣, p38␤1, p38␤2, and p38␥ were immunoprecipitated from cells co-transfected with empty vector (Ϫ) or activated MKK6 (ϩ) 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).

FIG. 3. Substrate specificity of p38 MAP kinases.
A, epitopetagged p38 MAP kinases were transfected in COS cells. The cells were then irradiated with 80 J of UV-C per m 2 (ϩ) or left untreated (Ϫ). 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. rylation by p38␤2 was less than p38␣ and p38␥. However, the fold-activation of p38␤2 activity was similar to that detected for p38␣ and p38␥ (Fig. 3A). These data indicate that the substrate specificity of these p38 MAP kinase isoforms was similar.
To further examine the substrate specificity of p38␤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 p38␣ MAP kinase, but not p38␤2 or p38␥, phosphorylated Mapkap-K2 (Fig. 3A). Thus, the p38␤2 MAP kinase substrate specificity differs from p38␣ MAP kinase.
The results of substrate analysis indicate that the substrate specificity of p38␤2 MAP kinase was similar to p38␥ (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 p38␤2 MAP kinase activity using the transcription factor ATF2 as the substrate. ATF2 was found to be a substrate for p38␣, p38␤2, and p38␥ MAP kinase (Fig. 3A). We have previously reported that p38␣ 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 p38␣ MAP kinase (Fig. 3B). In contrast, experiments using p38␥ 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 p38␥ MAP kinase, or that the phosphorylation of Thr-71 precedes the phosphorylation of other sites by p38␥ MAP kinase. Comparative studies using p38␤2 MAP kinase demonstrate that this MAP kinase isoform differs from both p38␣ and p38␥ 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 p38␤2 (Fig. 3B). These data suggest that both Thr-69 and Thr-71 are phosphorylated by p38␤2 MAP kinase. However, the phosphorylation of ATF2 by p38␤2 MAP kinase following replacement of both Thr-69 and Thr-71 with Ala residues indicate that p38␤2 phosphorylates a novel site on ATF2. Together, these data demonstrate that the substrate specificity of p38␤2 MAP kinase differs from the p38␣ and p38␥ MAP kinase isoforms.
Effect of a Pyridinyl Imidazole Drug on p38␤2 MAP Kinase Activity-We examined the effect of the pyridinyl imidazole derivative SB203580 (4, 5) on the protein kinase activity of p38␤2 MAP kinase. This drug has previously been shown to inhibit p38␣ MAP kinase activity (4,5). Here we demonstrate that SB203580 inhibits p38␤2 MAP kinase activity (Fig. 4). The dose response of inhibition of protein kinase activity was similar in experiments using p38␣ and p38␤2 MAP kinase (Fig.  4). In contrast, p38␥ MAP kinase was not inhibited by SB203580. The insensitivity of p38␥ 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 p38␣ and p38␤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 p38␤2 MAP kinase is therefore likely to be a physiologically relevant mediator of the p38 MAP kinase signal transduction pathway.
The p38␤2 MAP Kinase Is Activated by Proinflammatory Cytokines and Environmental Stress-The p38␣ and p38␥ MAP kinases are regulated by numerous extracellular stimuli, including proinflammatory cytokines and environmental stress (38). We compared the regulation of p38␤2 MAP kinase with the p38␣ and p38␥ MAP kinases. HeLa cells were transfected with epitope-tagged p38␣, p38␤2, and p38␥ 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-␣ and IL-1␣, environmental stress (UV irradiation and osmotic shock), and treatment with anisomycin (an inhibitor of protein synthesis) caused a marked increase in the activity of p38␤2 MAP kinase (Fig. 5). The strongest activation of p38␤2 was caused by the exposure of cells to UV radiation. Although ATF2 phosphorylation by p38␤2 was consistently less than that observed in kinase assays with p38␣ or p38␥, the foldincrease in protein kinase activity caused by UV radiation was similar for each p38 MAP kinase isoform (Fig. 5). These data demonstrate that p38␤2, like p38␣ and p38␥, is activated in vivo by a stress-induced signal transduction pathway.  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). using p38␣ and p38␥ MAP kinases. MKK3 caused strong activation of p38␣, a lower level of activation of p38␥, and did not activate p38␤2 (Fig. 6A). Similarly, MKK4 activated p38␣ strongly, weakly activated p38␥, and did not activate p38␤2 (Fig. 6B). In contrast, MKK6 caused strong activation of p38␣, p38␤2, and p38␥ MAP kinases (Fig. 6C). The effect of MKK3 and MKK4 to activate p38␣ and p38␥, but not p38␤2, indicates that the regulation of p38␤2 MAP kinase differs from the other p38 MAP kinases. The selective effect of MAP kinase kinases to regulate p38␤2 MAP kinase activity suggests that extracellular stimuli may selectively regulate p38 MAP kinase isoforms.

Selective Activation of p38 MAP Kinases by MAP Kinase
The p38␤2 MAP Kinase Is a Substrate for MKK6, but Not MKK3-The effect of MKK3 to activate p38␣ and p38␥ MAP kinases, but not p38␤2, could be accounted for by many mechanisms. One possibility is that p38␤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 p38␣ and p38␤2 MAP kinase, and caused a lower level of phosphorylation of p38␥ MAP kinase (Fig. 7B). In contrast, MKK3 caused a similar level of phosphorylation of p38␣ and p38␥ MAP kinases, but no phosphorylation of p38␤2 (Fig. 7A). These data demonstrate that MKK3 and MKK6 differentially phosphorylate p38 MAP kinase isoforms. Furthermore, these data indicate that p38␤2 MAP kinase is a substrate for MKK6, but not MKK3.
Transcriptional Regulation by the MKK6-p38␤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 p38␣ and p38␥ MAP kinases (Fig. 8, A and C). In contrast, p38␤2 MAP kinase did not increase MKK3-stimulated Elk-1 transcriptional activity (Fig.  8B). Control experiments using activated MKK6 demonstrated that p38␣, p38␤2, and p38␥ MAP kinases increased MKK6stimulated 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).
Together, these data indicate that MKK6 can couple to p38␣, p38␤2, and p38␥ MAP kinases to initiate a biological response, while MKK3 can couple only to p38␣ and p38␥ MAP kinases. These data are consistent with the potent activation of p38␤2 by MKK6 and the ineffective activation of p38␤2 by MKK3 in vitro and in vivo (Figs. 6 and 7). DISCUSSION The stress-activated MAP kinases include the JNK and p38 groups (3). The JNK group consists of 10 members that are FIG. 5. The p38␤2 MAP kinase is activated by pro-inflammatory cytokines and environmental stress. The activity of epitopetagged p38␣, p38␤2 and p38␥ MAP kinases was measured in immune complex protein kinase assays using [␥-32 P]ATP and ATF2 as substrate. The effect of treatment of Hela cells (30 min) with 10 ng/ml TNF-␣, 10 ng/ml IL-1␣, 300 mM sorbitol, 10 g/ml anisomycin, and 80 J of UV-C per m 2 was examined. The phosphorylated ATF2 was detected by autoradiography (15 min). To detect the lower level of p38␤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%). , and MKK6 (panel C) to activate p38␣, p38␤2, and p38␥ was tested in co-transfection assays. COS-7 cells were transfected with epitope-tagged p38␣, p38␤2, or p38␥ 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. 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 stressactivated MAP kinases consists of multiple isoforms (1). These isoforms include p38␣ (4,12,16,17,22), p38␤ (23), and p38␥ (24 -27). Recent studies indicate the presence of a fourth p38 MAP kinase isoform, p38␦ (28,29). In addition, alternatively spliced forms of p38␣ 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 (p38␤2) that is selectively activated by the MAP kinase kinase MKK6.
The p38␤2 Protein Kinase Is a Novel MAP Kinase-We report the molecular cloning of p38␤2 MAP kinase, a novel human stress-activated protein kinase. This enzyme is most similar to the previously characterized p38␤ MAP kinase (p38␤1) (23) and may be derived from the same gene by alternative splicing. The p38␤2 MAP kinase contains a 24-base pair deletion within the coding region of p38␤1 MAP kinase. This deletion represents a significant difference between these p38␤ MAP kinase isoforms. The p38␤1 MAP kinase contains an 8-amino acid insertion in the kinase domain that is absent in p38␤2 MAP kinase (Fig. 1).
It is most likely that p38␤1 and p38␤2 MAP kinases represent alternatively spliced forms of the same gene. However, sequence analysis of a p38␤2 MAP kinase genomic clone demonstrated that the site of the p38␤1 insertion was present within an exon (not at an exon boundary) and that the 24 base pair p38␤1 insertion sequence was not detected in the genomic clone (data not shown). These data suggest that p38␤1 and p38␤2 may be encoded by different genes. Alternatively, the insertion present in p38␤1 may be derived by post-transcriptional processing of the p38␤2 transcript. Further studies are required to resolve this issue.
Our studies of the origin of p38␤1 have been limited because we have been unable to detect p38␤1 by RT-PCR analysis of human mRNA. No difficulty was experienced in the detection of p38␤2. We interpret these data to indicate that p38␤2 is the major p38␤ MAP kinase isoform that is expressed in many human tissues. The expression of the p38␤1 isoform may be restricted to specific tissues.
Intriguingly, we have not been able to detect any protein kinase activity in experiments using the p38␤1 MAP kinase. Similar negative data were obtained in vitro and in vivo (Fig.  2). The lack of activity of p38␤1 is in contradiction with studies by Jiang et al. (23) who reported that p38␤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 p38␤1 in vitro (Fig.  1A), nor have we been able to activate transfected p38␤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 p38␣ and p38␤ 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 p38␣, based on the effects of pyridinyl imidazole drugs, could be mediated by p38␤2 MAP kinase. Therefore, p38␤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).
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 spe- 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 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 FASinduced 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 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 ␤-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 ␤-galactosidase expression. The relative luciferase activity detected following co-transfection of the empty vector, activated MKK3, or activated MKK6 together with p38␣ (panel A), p38␤2 (panel B), or p38␥ (panel C) is presented. D, effect of p38 MAP kinase isoforms 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 p38␣, p38␤2, and p38␥. MKK3 activates p38␣ and p38␥, but not p38␤2. The MAP kinase kinase MKK4 activates p38␣ and does not activate p38␤2. MKK4 is also a weak activator p38␥. The differential activation of these p38 MAP kinase isoforms may lead to cell type-specific and stimulusspecific cellular responses.
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 (Ϫ) or activated MKK6 (ϩ). 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 cotransfected with Elk-1 and p38 MAP kinase isoforms with empty vector (Ϫ) or activated MKK6 (ϩ). 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). 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 p38␣, p38␤2, and p38␥ MAP kinases, while MKK3 targets p38␣ and p38␥ MAP kinases. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinase isoforms.