Murine p38-δ Mitogen-activated Protein Kinase, a Developmentally Regulated Protein Kinase That Is Activated by Stress and Proinflammatory Cytokines*

The p38 mitogen-activated protein kinases (MAPK) play a crucial role in stress and inflammatory responses and are also involved in activation of the human immunodeficiency virus gene expression. We have isolated the murine cDNA clones encoding p38-δ MAPK, and we have localized the p38-δ gene to mouse chromosome 17A3-B and human chromosome 6p21.3. By using Northern andin situ hybridization, we have examined the expression of p38-δ in the mouse adult tissues and embryos. p38-δ was expressed primarily in the lung, testis, kidney, and gut epithelium in the adult tissues. Although p38-δ was expressed predominantly in the developing gut and the septum transversum in the mouse embryo at 9.5 days, its expression began to be expanded to many specific tissues in the 12.5-day embryo. At 15.5 days, p38-δ was expressed virtually in most developing epithelia in embryos, suggesting that p38-δ is a developmentally regulated MAPK. Interestingly, p38-δ and p38-α were similar serine/threonine kinases but differed in substrate specificity. Overall, p38-δ resembles p38-γ, whereas p38-β resembles p38-α. Moreover, p38-δ is activated by environmental stress, extracellular stimulants, and MAPK kinase-3, -4, -6, and -7, suggesting that p38-δ is a unique stress-responsive protein kinase.

The first p38 MAPK (hereafter designated as p38-␣) was identified initially in lipopolysaccharide-stimulated macrophages and was found later to share significant homology with the yeast HOG1 kinase (7,41). Subsequently, the human p38-␣ homologues (CSBPs) were isolated by using radiolabeled and radiophotoaffinity labeled pyridinyl imidazole compounds, which block inflammatory cytokine biosynthesis by monocytes stimulated with lipopolysaccharide (8). Another member (p38-␤) of the p38 MAPK family was identified and cloned, which is very homologous (with 75% amino acid identity) to p38-␣ (42). The third member (p38-␥) of the p38 MAPK family was recently isolated as ERK-6 (43) and SAPK3 (44), which share significant homology (63% amino acid identity) with p38-␣. All these p38 MAPK members contain a characteristic Thr-Gly-Tyr motif within the kinase subdomain VIII.
Here, we present a murine p38 MAPK family member, p38-␦, * This work was supported by Amgen, Inc. (to M. C.-T. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF092534, AF092535, X79483, D83073, U66243, L35264.

EXPERIMENTAL PROCEDURES
Isolation of Murine and Human P38-␦ cDNAs-A rat 1.3-kilobase pair expressed sequence tag (EST) cDNA clone with ϳ62% homology to mouse p38-␤ (GenBank TM accession number D83073) cDNA was used as a probe to screen a rat lung cDNA library in gt11 phage vector (CLONTECH Laboratories). For hybridization, replicate filters were prehybridized for 1 h at 68°C in Express hybridization buffer (CLON-TECH Laboratories) and hybridized 12 h at 68°C in the same solution with the [ 32 P]dCTP-labeled probe. After hybridization, the filters were washed several times at high stringency, at 65°C in 0.1% SDS, 0.2ϫ SSC (1ϫ SSC, 150 mM NaCl and 15 mM sodium citrate), and subjected to autoradiography. Several positive clones were picked and purified after screening 4 ϫ 10 6 phages. The cDNA inserts of these positive phage clones were subsequently subcloned into pCR3.1 plasmid vector (Invitrogen). After analysis of the inserts, the longest cDNA clone was sequenced on both strands, using a PCR procedure employing fluorescent dideoxynucleotides and a model 373A automated sequencer (Applied Biosystems). Similarly, for human p38-␦ cDNA cloning, the same EST cDNA probe was used to screen a human lung cDNA library in TripEx phage vector (CLONTECH Laboratories). Several positive clones were obtained, and the cDNA inserts of these phage clones were converted in vivo into pTripEx plasmid vector, according to the manufacturer's instructions. A candidate full-length cDNA clone was sequenced on both strands as described above. Sequence comparisons were aligned with the Bestfit program of the GCG sequence analysis software package (Wisconsin Package version 9.0).
Northern Blot Analysis-Poly(A) ϩ RNAs from various mouse tissues were obtained from CLONTECH Laboratories. Each sample (2 g) was denatured and electrophoresed on a 1.2% agarose gel containing formaldehyde and then transferred to a Hybond-N membrane (Amersham Pharmacia Biotech) in 20ϫ SSC as described (46). Murine p38-␦ or human ␤-actin cDNA was labeled with [ 32 P]dCTP to a specific activity of approximately 10 8 dpm/g. Membranes were hybridized with either the p38-␦ or ␤-actin cDNA probe, then washed at high stringency, at 65°C in 0.2ϫ SSC, 0.1% SDS, and subjected to autoradiography. Probes were removed in 0.5% SDS at 95-100°C.
Briefly, fetuses and tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline overnight, dehydrated, and infiltrated with paraffin. Serial sections at thickness of 5-7 m were mounted on gelatin-coated slides, deparaffinized in xylene, rehydrated, and postfixed. The tissue sections were digested with proteinase K, post-fixed, treated with triethanolamine/acetic anhydride, washed, and dehydrated. The cRNA transcripts were synthesized from linearized cDNA templates to generate antisense and sense probes, according to manufacturer's conditions (Ambion) and labeled with 35 S-UTP (Ͼ1000 Ci/ mmol; Amersham Pharmacia Biotech). cRNA transcripts larger than 200 nucleotides were subjected to alkali hydrolysis to give a mean size of 70 nucleotides. The tissue slides were hybridized overnight at 52°C in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl, pH 7.4, 10 mM NaPO 4 , 5 mM EDTA, 10% dextran sulfate, 1ϫ Denhardt's, 50 g/ml total yeast RNA, and 5-7.5 ϫ 10 4 cpm/l 35 S-labeled cRNA probe. The tissue slides were subjected to stringent washing at 65°C in 50% formamide, 2ϫ SSC, 10 mM dithiothreitol, and washed in phosphatebuffered saline before treatment with 20 g/ml RNase A at 37°C for 30 min. Following washes in 2ϫ SSC and 0.1ϫ SSC at 37°C for 10 min, the slides were dehydrated and dipped in Kodak NTB-2 nuclear track emulsion and exposed for 2-3 weeks in light-tight boxes with desiccant at 4°C. Photographic development was carried out in Kodak D-19. The tissue slides were counterstained lightly with toluidine blue and analyzed using both light and dark field optics of a microscope. Sense control cRNA probes indicate the background levels of the hybridization signal.
Cell Culture and Transfections-293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Cells to be transfected were plated the day before transfection at a density of 2 ϫ 10 6 cells per 100-mm dish. 293T cells were co-transfected with expression plasmids (10 g each plasmid per dish) as indicated with pVA1 (10 g per dish) to enhance transient protein expression, using the calcium phosphate precipitation protocol (Specialty Media, Inc.). The transfected 293T cells were harvested 48 h after transfection. For cell stimulation, 293T cells were treated with human TNF-␣ (20 ng/ml) for 10 min before harvest.
Phosphoamino Acid Analysis-The phosphorylated proteins obtained from immunocomplex kinase assays were transferred electrophoretically to polyvinylidene difluoride membranes. The spots containing phosphoproteins on the membranes were excised according to the bands on autoradiograms and then hydrolyzed in 50 l of 6 N HCl for 1 h at 110°C. The supernatant was lyophilized and dissolved in 6 l of pH 1.9 buffer (2.2% formic acid and 7.8% acetic acid) containing cold phosphoamino acids as markers. The phosphoamino acids were resolved electrophoretically in two dimensions using a thin layer cellulose (TLC) plate with two pH systems as described (49). The markers were visualized by staining with 0.2% ninhydrin in acetone, and the 32 P-labeled residues were detected by autoradiography.
Lymphocyte Culture and Microscope Slides Preparation-Lymphocytes isolated from human blood were cultured in ␣-minimal essential medium supplemented with 10% fetal bovine serum and phytohemagglutinin at 37°C for 68 -72 h. The lymphocyte cultures were treated with bromodeoxyuridine (0.18 mg/ml, Sigma) to synchronize the cell population. The synchronized cells were washed three times with serum-free medium to release the block and recultured at 37°C for 6 h in ␣-minimal essential medium with thymidine (2.5 g/ml, Sigma). The cells were harvested, and the cell slides were prepared by using standard procedures including hypotonic treatment, fixation, and air-drying.
Chromosome Mapping by Fluorescence in Situ Hybridization (FISH)-The procedure for FISH detection was performed as described previously (50,51). Briefly, the cell slides were baked at 55°C for 1 h. After RNase treatment, the slides were denatured in 70% formamide in 2ϫ SSC for 2 min at 70°C followed by dehydration with ethanol. DNA probes were labeled with biotinylated dATP at 15°C for 1 h, using the Life Technologies, Inc., BioNick labeling kit (Life Technologies, Inc.). Probes were denatured at 75°C for 5 min in a hybridization buffer containing 50% formamide and 10% dextran sulfate and loaded onto the denatured chromosomal slides. After 16 -20 h hybridization, the slides were washed and incubated with fluorescein isothiocyanate-conjugated avidin (Vector Laboratories), and the signal was amplified as described (51). FISH signals and the 4Ј,6Ј-diamidino-2-phenylindole (DAPI) banding patterns were recorded separately by taking photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with the DAPI-banded chromosomes (52).

Molecular Cloning and Structure of Murine and Human
P38-␦ cDNAs-A 1328-bp partial cDNA sequence with high homology (ϳ62% amino acid identity) to the kinase domain of mouse p38-␤ cDNA was identified from the Amgen EST data base of a rat colon cDNA library. Initially, we termed this cDNA an IKK-like kinase. By using this rat cDNA as a probe, we have isolated a putative full-length cDNA clone from a rat lung cDNA library. The nucleotide sequence of 1577 bp contains a single open reading frame of 1098 bp encoding a FIG. 1. Nucleotide and amino acid sequences of murine p38-␦ cDNA and sequence alignment. A, the nucleotide and predicted amino acid sequences of murine p38-␦ are shown. The predicted amino acid sequence is indicated below the first nucleotide of each codon, and the termination codon is marked with an asterisk. The polyadenylation signal is underlined. GenBank TM accession numbers for murine and human p38-␦ are AF092534 and AF092535, respectively. B, alignment of the deduced amino acid sequences of murine and human p38-␦ (mp38-␦ and hp38-␦) with those of human p38-␥ (hp38-␥, GenBank TM accession number X79483), murine and human p38-␤ (mp38-␤ and hp38-␤, GenBank TM accession numbers D83073 and U66243, respectively), and human p38-␣ (hp38-␣, GenBank TM accession number L35264). The sequences (single letter codes) were aligned with the Bestfit program of the GCG sequence analysis software package. Gaps were introduced to obtain optimal alignment and are denoted by dashes. Identical amino acids among at least five proteins are highlighted with solid boxes. Roman numerals on the top line denote the 12 conserved kinase subdomains identified by Hanks and Quinn (55). The bottom consensus sequence indicates amino acids that are invariant (uppercase) or almost invariant (lowercase) in a comparison of the catalytic domains of 100 Ser/Thr protein kinases (56). The asterisks highlight the fully conserved TGY motif within the kinase subdomain VIII. polypeptide of 366 amino acids, and followed by a 471-bp 3Јuntranslated region that contains the polyadenylation signal at position 1512 (Fig. 1A). The calculated molecular mass of the deduced amino acid sequence is about 41 kDa. A homology search of the GenBank TM data base revealed that the coding sequence of this clone is very similar with those of p38-␣ (8), p38-␤ (42), and p38-␥ (43,44), and designated as murine p38-␦.
By using the murine p38-␦ cDNA as a probe, we have also isolated a putative full-length cDNA clone from a human lung cDNA library. The nucleotide sequence of 1794 bp contains a single open reading frame of 1095 bp encoding a polypeptide of 365 amino acids and followed by a 678-bp 3Ј-untranslated region that contains the polyadenylation signal at position 1749 (data not shown). Sequence alignments showed that the deduced amino acid sequences of human and murine p38-␦ exhibit 92% identity, and p38-␦ is approximately 63, 61, and 67% identical to p38-␣, p38-␤, and p38-␥, respectively (Fig. 1B). The putative dual phosphorylation TGY motif within the kinase subdomain VIIIis fully conserved among the known mammalian p38 family members.
Expression of P38-␦ Is Regulated in Different Developmental Stages-The expression of p38-␦ was examined in a variety of mouse adult tissues by Northern blot analysis. A tissue Northern blot was probed with the murine p38-␦ cDNA, and a major p38-␦ transcript (ϳ3 kilobase pair) was identified in the lung, testis, kidney, and at lower levels in the liver and skeletal muscle (Fig. 2). Furthermore, we examined the expression of p38-␦ mRNA in various days of mouse embryos and adult tissues by in situ hybridization using a 35 S-labeled antisense p38-␦ RNA probe, followed by autoradiography. Whereas p38-␦ was expressed predominantly in the developing gut and the septum transversum in the mouse embryo at 9.5 days (Fig. 3A), its expression began to be localized to the gut, heart ventricle, neuroepithelium of the fourth ventricle of the brain, cochlea, and semicircular canal of the inner ear and oropharynx in the 12.5-day embryo (Fig. 3C). At 15.5 days, the expression of p38-␦ was further expanded to the adrenal gland, duodenum, intestine, epidermis, kidney, and lung thalamus (Fig. 3, D and E). p38-␦ was expressed virtually in most developing epithelia in embryos, suggesting that p38-␦ is a developmentally regulated MAPK that may play a role in embryonic development. In the adult mouse, significant p38-␦ signal was detected in the lung, liver, testis, skeletal muscle, and gut epithelium in the adult tissues (data not shown). The negative control hybridization with a 35 S-labeled sense p38-␦ RNA probe showed the level of background in a sagittal section of a 9.5-day embryo (Fig. 3B) and a 15.5-day embryo (Fig. 3F). Taken together, these results indicate that the p38-␦ mRNA expression was modulated in different developmental stages, suggesting that p38-␦ is a developmentally regulated MAP kinase.
The P38-␦ Gene Is Localized to Mouse Chromosome 17A3-B and Human Chromosome 6p21.3-To determine the chromosomal localization of the p38-␦ gene in the mouse genome, the biotinylated murine p38-␦ cDNA probe was used to map the mouse chromosome, using the fluorescence in situ hybridization (FISH) technique (50,51). A specific region of one chromosome showed the FISH positive with the p38-␦ probe (Fig. 4A). Under the condition used, the hybridization efficiency was approximately 65% for this probe (among 100 checked mitotic figures, 65 of them revealed positive signals on one pair of the chromosomes). Since the DAPI banding was used to identify the specific chromosome, the assignment between signal from the probe and the mouse chromosome 17 was established (Fig.  4B). The detailed position was further determined to region A3-B based on the summary of 10 photographs. There was no

. In situ hybridization (ISH) analysis of murine p38-␦ mRNA expression in mouse embryos tissues.
A, ISH of a sagittal section of a 9.5-day embryo (5 ϫ magnification) shows strong expression of p38-␦ in the primitive foregut (a), septum transversum, which is the future site of liver development (b), and the ventricle of the primitive heart (c). B, ISH using a sense control probe shows the level of background in the same sagittal section as described in A (5 ϫ magnification). C, ISH of a frontal section of a 12.5-day embryo (2.5 ϫ magnification). Intense labeling is observed in the gut (d), and significant signals are found in the heart ventricle (e), the neuroepithelium of the fourth ventricle of the brain (f and g), the cochlea of the inner ear (h), the semicircular canal of the inner ear (i), and the oropharynx (j). D, ISH of a sagittal section of a 15.5-day embryo abdomen (2.5 ϫ magnification). Labeling is observed in the adrenal gland (k), the duodenum (l), the kidney (m), the small intestine (n), the large intestine (o), and the epidermis (p). E, ISH of a sagittal section of a 15.5-day embryo abdomen and thorax (2.5 ϫ magnification). Strong signals are found in the epidermis (q), the intestine (r), the lung (s), and the kidney (t). F, ISH using a sense control probe shows the level of background in the same sagittal section as described in E (2.5 ϫ magnification).
other positive locus detectable under the condition used; therefore, the gene of p38-␦ was mapped to mouse chromosome 17, region A3-B (Fig. 4C).
Similarly, the biotinylated human p38-␦ cDNA probe was used to map the human chromosome. A specific region of one chromosome showed the FISH positive with the p38-␦ probe (Fig. 5A), and the hybridization efficiency was approximately 70% for this probe. The assignment between signal from the probe and the short arm of chromosome 6 was established (Fig.  5B), and the detailed position was further determined to region p21.3. Since there were no other positive loci detected under the condition used, the gene of p38-␦ was localized to human chromosome 6, region p21.3 (Fig. 5C).
The Dual Phosphorylation TGY Motif Is Essential for Activation of P38-␦-Since activation of p38 MAPK is mediated by dual phosphorylation on Thr 180 and Tyr 182 within the kinase domain (17), we investigated whether this Thr 180 -Gly-Tyr 182 motif was essential for p38-␦ activation. A mutant p38-␦ was constructed by substituting the Thr 180 -Gly-Tyr 182 motif with Ala 180 -Gly-Phe 182 by site-directed mutagenesis, p38-␦(AGF) mutant, and examined in the co-transfection and stimulation experiments as described above. Unlike the wild-type p38-␦, co-transfection of 293T cells with the p38-␦(AGF) mutant plus MKK-3 or MKK-6 failed to activate the p38-␦ kinase activity (Fig. 7B, lanes 7 and 8), and this p38-␦(AGF) mutant could not respond to H 2 O 2 stimulation (Fig. 7B, lane 9). Western blot analysis indicated that the p38-␦(AGF) mutant was expressed at similar levels as the wild-type p38-␦ in this experiment (bottom panel). Taken together, these results suggest that the dual phosphorylation TGY motif is required for p38-␦ activation. DISCUSSION We have identified and isolated a novel murine and human p38 MAPK family member, p38-␦, whose sequence is most similar to p38-␥ (67% amino acid identity) among the p38 family members, whereas p38-␣ is most homologous to p38-␤ (75% amino acid identity). It is intriguing that expression of p38-␦ was primarily in the developing gut and the septum transversum in the early mouse embryo (at 9.5 days) initially, then its expression began to be expanded to many specific tissues of the later embryo (at 12.5 days). At 15.5 days, p38-␦ was expressed virtually in most developing epithelia in embryos, suggesting that p38-␦ is a developmentally regulated MAPK that may play a role in embryonic development. Since all four p38 MAPK family members are closely related in structure and function, it is possible that expression of p38-␣, -␤, and -␥ may also be regulated in different developmental stages. They may play important roles in stress and inflammatory responses in different tissues during embryonic development.
We elected to show different sectional perspectives for embryos at days 9.5, 12.5, and 15.5, rather than presenting a common sectional perspective because different sectioning orientations are advantageous to view certain developing tissues that grow at different rates and change orientation as development proceeds. Additionally, different sectioning orientations provide depths of the labeling for a more comprehensive study. Interestingly, expression of p38-␦ correlated with areas of epithelial development in the gut, kidney, adrenal, lung, and skin. The p38-␦ mRNA expression was also detected in some neurons that are derived from the ectoderm. Overall, the pattern of expression suggests that p38-␦ is expressed in the FIG. 6. p38-␦ and p38-␣ are serine/threonine kinases but differ in substrate specificity. 293T cells were transfected with either the Flag-tagged human p38-␣ or the Flag-tagged murine p38-␦ cDNA (10 g each). pVA1 plasmid (10 g) containing adenovirus VA1 RNA gene was also included in each transfection to enhance transient protein expression. The cells were harvested 48 h after transfection without stimulation. After immunoprecipitation with anti-Flag M2 mAb, p38-␣ (A) or p38-␦ (B) kinase activity was measured by immunocomplex kinase assays in the presence of the indicated substrates. The in vitro phosphorylated ATF-2-(1-96) by p38-␣ (C) or p38-␦ (D) were gel-isolated, and phosphoamino acids were analyzed electrophoretically in two dimensions using a TLC with two pH systems. The relative positions of unlabeled phosphoamino acids are indicated below the autoradiographs. S, serine; T, threonine; Y, tyrosine.
proliferating and nonproliferating layers of epithelia.
Since the kinase domains of all four p38 family members are very conserved, it is of interest to test whether they share the same substrate specificity. It has been shown that p38-␣ and p38-␤ phosphorylate similar substrates including ATF-2, PHAS-1, and MAPKAP kinases (42). Although p38-␥ can also phosphorylate ATF-2, it cannot phosphorylate MAPKAP kinases effectively (44). Here, we showed that p38-␦ phosphorylated ATF-2 and PHAS-1 strongly but not MAPKAP kinase-2 which is a physiological substrate for p38-␣. This result suggests that p38-␦ shares substrate specificity with p38-␥. Thus, in terms of sequence similarity and substrate specificity, p38-␦ most resembles p38-␥, whereas p38-␤ most resembles p38-␣. In addition, p38-␦ differs from p38-␣ in phosphorylation specificity against serine residues. We showed that p38-␣ phosphorylated serine and threonine, whereas p38-␦ phosphorylated threonine predominantly, suggesting that p38-␦ is primarily a threonine kinase and is dissimilar with p38-␣ in phosphorylation specificity against serine residues.
It has been shown that p38 MAPKs are activated by dual phosphorylation at the Thr 180 -X-Tyr 182 motif within the kinase subdomain VIII (17). P38-␦ contains the dual phosphorylation TGY motif that is fully conserved among all four p38 MAPK family members. Mutation of the Thr 180 and Tyr 182 residues in this TGY motif abrogated the p38-␦ kinase activity and its activation by extracellular stimuli or upstream kinases (MKKs). Therefore, the dual phosphorylation TGY motif is indispensable for the kinase activity and activation of p38-␦. Similar to other p38 MAPKs, p38-␦ is activated by environmental stress and proinflammatory cytokines. This activation is presumably regulated by dual phosphorylation on Thr 180 and Tyr 182 . P38-␦ is also activated by its upstream kinases (MKKs) which may phosphorylate Thr 180 and/or Tyr 182 in the TGY motif. However, it is unclear whether all these upstream kinases (MKK-3, -4, -6, and -7) phosphorylate the TGY motif of p38-␦ in the same manner.
It has been shown that p38-␣ is preferably activated by MKK-3 in PC-12 cells, whereas p38-␣ is predominantly acti-vated by MKK-6 in monocytes and KB cells, suggesting that p38-␣ is activated by different MKKs in a cell type-dependent manner (44). Since MKK-1, -2, and -5 are specific activators for ERKs (24,25,34,35), we examined the other MKKs on p38-␦ activation. Unlike p38-␣, we found that p38-␦ was activated by MKK-3, -4, -6, and -7 approximately equally well in 293T cells, suggesting that the regulation of p38-␦ may be distinct from p38-␣. However, it is still unknown whether or not the regulation of p38-␦ depends on cell type. Additionally, p38-␦ was activated in response to a variety of stimulants including environmental stress, TNF-␣, IL-1␣, and EGF. Although most of these factors stimulated p38-␦ to a relatively similar degree in 293T cells, it is possible that the kinase reaction was not in a linear range and ATF-2 might not be the physiological substrate for p38-␦. Therefore, the degrees of stimulation of these factors may not reflect their physiological effects on p38-␦ in vivo. Although activation of p38-␦ by EGF may be somewhat surprising, it has been shown recently that p38 MAPK (p38-␣) can be stimulated by EGF in certain cell types (53,54). Thus, further investigation of these factors is required to understand fully the physiological activators of p38-␦.
p38-␣ (CSBP) has been implicated in the regulation of inflammatory cytokine biosynthesis through the use of specific p38-␣ inhibitors (8). We examined one of the p38-␣ inhibitors in our phosphorylation studies and showed that it was ineffective in blocking p38-␦ activity. Further investigation of other compounds that may inhibit p38-␦ function is necessary to understand whether p38-␦ is involved in the regulation of inflammatory cytokine production in cells.
We showed that the gene of p38-␦ was localized to mouse chromosome 17 region A3-B and human chromosome 6p21.3. Interestingly, the gene of p38-␣ (CSBP) has also been mapped to human chromosome 6p21.3/21.2 (52). To our knowledge, this is the first description of chromosomal localization of p38-␦. At present, it is unknown whether mutation or defect of p38-␦ gene is involved in any diseases. Mutation analysis in mice or human with monogenic disorders that map to mouse chromosome 17A3-B or human chromosome 6p21.3 will evaluate the FIG. 7. p38-␦ is activated by environmental stress, extracellular stimulants, and MAPK kinase-3, -4, -6, and -7. A, 293T cells were transfected with the the Flag-tagged murine p38-␦ cDNA (10 g each) in the absence or presence of extracellular stimuli as indicated. pVA1 plasmid (10 g) containing adenovirus VA1 RNA gene was also included in each transfection to enhance transient protein expression. The cells were harvested 48 h after transfection without stimulation. After immunoprecipitation with anti-Flag M2 mAb, p38-␦ kinase activity was measured by immunocomplex kinase assays, using ATF-2-(1-96) as a substrate. As a control for p38-␦ expression, equal amounts of cell lysate (200 involvement of this gene in diseases. Furthermore, targeted disruption (knock-out) of this gene in mice may provide evidence for the relationship between its function and diseases.
In summary, we have isolated and characterized p38-␦ and determined its global tissue distribution, chromosomal localization, and its biological activity. Further investigation of the regulation of p38-␦ may contribute to a better understanding of the roles that p38-␦ have in normal development and pathological processes.