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INTRODUCTION |
The mitogen-activated protein kinase
(MAPK)1 cascade is a major
signaling system by which cells transduce extracellular stimuli into
intracellular signals to control the expression of genes essential for
cellular processes such as cell proliferation, differentiation, and
stress responses (1, 2). In mammalian cells, these kinases include
extracellular signal-regulated protein kinases (ERKs) (3), the c-Jun
amino-terminal kinases (JNK)/stress-activated protein kinases (SAPKs)
(4-6), and p38 MAPKs (7-10). Whereas ERKs are activated rapidly in
response to the binding of growth factors to growth factor receptors
(11, 12), JNKs/SAPKs and p38 MAPKs are stimulated by environmental
stress (i.e. osmotic shock, ultraviolet irradiation,
cytotoxic chemicals, etc.) and proinflammatory cytokines
(i.e. interleukin-1 (IL-1) and tumor necrosis factor (TNF))
(4, 5, 7, 8, 13-17). Although the physiological function of the ERKs
and JNKs/SAPKs in signal transduction pathways has been extensively
studied (1, 3, 18-21), the functional role of the p38 MAPK signaling
pathway is relatively less understood (7-9, 17, 22). Nevertheless, p38 MAPKs play an important role in stress and inflammatory responses and
are also involved in activation of the human immunodeficiency virus
type 1 promoter (23).
These MAP kinases have the unique feature of being activated by
phosphorylation on threonine (Thr) and tyrosine (Tyr) residues by
upstream dual-specificity kinases, i.e. MAP kinase kinases (MKKs or MEKs) (18, 20). This dual phosphorylation Thr-X-Tyr motif is located within the kinase subdomain VIII where ERK is Thr-Glu-Tyr; JNK/SAPK is Thr-Pro-Tyr; and p38 MAPK is Thr-Gly-Tyr. MKK-1 and MKK-2 phosphorylate and activate ERK-1 and ERK-2 (24, 25),
whereas MKK-3 and MKK-6 activate p38 MAPK specifically (26-32).
Although MKK-4 (SEK-1) stimulates JNK/SAPK and p38 MAPK (26, 27, 33),
MKK-5 phosphorylates and activates ERK-5 (34, 35). Recently, MKK-7 has
been shown to activate JNKs/SAPKs specifically but not p38 MAPKs and
ERKs (36). Each MAP kinase group has a unique substrate specificity and
is regulated by a distinct signal transduction pathway (18, 20, 21).
For instance, ERK-1 and ERK-2 phosphorylate and activate the
transcription factor Elk-1 (37, 38). JNKs/SAPKs phosphorylate and
activate the transcription factors c-Jun (4, 5), ATF-2 (39), and Elk-1
(40). The p38 MAPKs phosphorylate and activate the transcription
factors ATF-2 (17, 26) and Elk-1 (29).
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-
, whose
sequence is significantly homologous to p38-
(63% amino acid identity). We showed that expression of p38-
mRNA was regulated in different developmental stages, suggesting that p38-
is a developmentally regulated MAPK. We characterized p38-
by determining its chromosomal location, stimulation by extracellular stimuli, and
activation by upstream kinases (MKKs). Moreover, we compared substrate
specificity and inhibitor sensitivity between p38-
and p38-
, and
we showed that they are discrete. Our results indicate that p38-
is
a unique stress-responsive protein kinase.
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EXPERIMENTAL PROCEDURES |
Isolation of Murine and Human P38-
cDNAs--
A rat
1.3-kilobase pair expressed sequence tag (EST) cDNA clone with
~62% homology to mouse p38-
(GenBankTM 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
(CLONTECH Laboratories) and hybridized 12 h at
68 °C in the same solution with the [32P]dCTP-labeled
probe. After hybridization, the filters were washed several times at
high stringency, at 65 °C in 0.1% SDS, 0.2× SSC (1× SSC, 150 mM NaCl and 15 mM sodium citrate), and
subjected to autoradiography. Several positive clones were picked and
purified after screening 4 × 106 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).
DNA, Protein, and Chemical Reagents--
The Flag-tagged p38-
expression plasmid was constructed from the murine p38-
cDNA by
the PCR technique using oligonucleotides 5'-AAGCTTGTCGACGCCACCATGGATTATAAAGATGATGATGATAAAAGCCTCATTCGGAAAAGGGGCTTC-3' and 5'-TATTGCGGCCGCTTATCACTGCAGCTTCATCCCACTTCG-3' as primers to incorporate a Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) at the
amino terminus of p38-
. The PCR-generated product was cloned into
the expression vector pCR3.1 plasmid vector (Invitrogen) and designated
pFlag-p38-
. The Flag-tagged p38-
(AGF) mutant expression plasmid
was generated from pFlag-p38-
plasmid by the PCR technique using
oligonucleotides 5'-GATGCGGAGATGGCTGGCTTTGTGGTGACCCGC-3' and
5'-GCGGGTCACCACAAAGCCAGCCATCTCCGCATC-3' as primers to replace the
Thr180-Gly-Tyr182 motif with
Ala180-Gly-Phe182, using a
QuickChangeTM site-directed mutagenesis kit (Stratagene).
The sequences of these cDNA constructs were confirmed by DNA
sequencing on both strands as described. The pVA1 (containing the
adenovirus VA1 RNA gene) plasmid was obtained as described previously
(45). The Flag-tagged p38-
, MKK-3, MKK-4, MKK-6, MKK-7, and MEK
kinase-5 (ASK-1) expression plasmids were kindly provided by Dr. R. Geronsin (Amgen Inc.). ATF-2-(1-96) and GST-MAPKAP kinase-2 were
purchased from Santa Cruz Biotechnology and Upstate Biotechnology Inc., respectively. PHAS-1 and p38-
inhibitor were purchased from
Stratagene. Myelin basic protein (MBP), anisomycin, and
Na3VO4 were purchased from Sigma. GST-c-Jun was
prepared as described previously (45), and anti-Flag M2 mAb was
purchased from Kodak Scientific Imaging Systems. Human TNF-
,
IL-1
, and epidermal growth factor (EGF) were purchased from R & D Systems.
Northern Blot Analysis--
Poly(A)+ RNAs from
various mouse tissues were obtained from CLONTECH
Laboratories. Each sample (2 µg) was denatured and electrophoresed on
a 1.2% agarose gel containing formaldehyde and then transferred to a
Hybond-N membrane (Amersham Pharmacia Biotech) in 20× SSC as described
(46). Murine p38-
or human
-actin cDNA was labeled with
[32P]dCTP to a specific activity of approximately
108 dpm/µg. Membranes were hybridized with either the
p38-
or
-actin cDNA probe, then washed at high stringency, at
65 °C in 0.2× SSC, 0.1% SDS, and subjected to autoradiography.
Probes were removed in 0.5% SDS at 95-100 °C.
In Situ Hybridization (ISH)--
ISH was performed as described
(47). 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 µm were mounted
on gelatin-coated slides, deparaffinized in xylene, rehydrated, and
post-fixed. 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
35S-UTP (>1000 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 NaPO4, 5 mM EDTA, 10% dextran sulfate, 1× Denhardt's, 50 µg/ml total yeast RNA, and 5-7.5 × 104 cpm/µl 35S-labeled cRNA probe. The
tissue slides were subjected to stringent washing at 65 °C in 50%
formamide, 2× SSC, 10 mM dithiothreitol, and washed in
phosphate-buffered saline before treatment with 20 µg/ml RNase A at
37 °C for 30 min. Following washes in 2× SSC and 0.1× 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 × 106 cells per 100-mm dish. 293T cells were co-transfected
with expression plasmids (10 µg each plasmid per dish) as indicated
with pVA1 (10 µg 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.
Immunoprecipitation and Western Blot Analysis--
Cells were
lysed in WCE lysis buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM
-glycerophosphate, 1% Triton
X-100, 10% glycerol, 1 mM dithiothreitol, 2 µg/ml
leupeptin, 5 µg/ml aprotinin, 1 mM Pefabloc (Boehringer
Mannheim) or phenylmethylsulfonyl fluoride, 1 mM sodium
orthovanadate). Soluble lysates were prepared by centrifugation at
10,000 × g for 30 min at 4 °C. The lysates were
precleared using Pansorbin cells (Calbiochem) and then incubated with
specific antibodies. After 16 h of incubation, immunocomplexes
were recovered with the aid of Gamma-Bind Sepharose beads (Amersham
Pharmacia Biotech) and then washed four times with lysis buffer.
Subsequently, immunoprecipitates were analyzed by Western blotting
after SDS-PAGE (10%), electroblotted onto polyvinylidene difluoride
membranes (Novex, Inc.), and probed with the corresponding rabbit
antiserum or mouse monoclonal antibody. Immunocomplexes were visualized by enhanced chemiluminescence (ECL) detection (Amersham Pharmacia Biotech) using goat anti-rabbit or anti-mouse antisera conjugated to
horseradish peroxidase as a secondary antibody (Pierce).
Immunocomplex Kinase Assays--
Immunocomplex kinase assays
were carried out as described previously (48). Specifically, cellular
p38-
or p38-
proteins were immunoprecipitated by incubation with
anti-Flag mAb and protein A-agarose beads (Bio-Rad) in WCE lysis
buffer. After 3 h of incubation at 4 °C, the immunoprecipitates
were collected and washed twice with WCE lysis buffer, twice with LiCl
buffer (500 mM LiCl, 100 mM Tris-Cl, pH 7.6, and 0.1% Triton X-100), and twice with kinase buffer (20 mM Mops, pH 7.6, 2 mM EGTA, 10 mM
MgCl2, 1 mM dithiothreitol, 0.1% Triton X-100,
and 1 mM Na3VO4). Pellets were then
mixed with 5 µg of substrate, 20 µCi of [
-32P]ATP,
and 15 µM unlabeled ATP in 30 µl of kinase buffer. The substrates included MBP, GST-c-Jun, GST-MAPKAP kinase-2, PHAS-1, and
ATF-2-(1-96) in the absence or presence of p38-
inhibitor (SB203580). The kinase reaction was performed for 30 min at
30 °C and terminated by boiling in an equal volume of Laemmli sample buffer, and the products were resolved by SDS-PAGE (10%). The gel was
dried and subjected to autoradiography.
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 µl of 6 N HCl for 1 h at 110 °C. The supernatant was
lyophilized and dissolved in 6 µl 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 32P-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 µg/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 2× 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 4',6'-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).
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RESULTS |
Molecular Cloning and Structure of Murine and Human P38-
cDNAs--
A 1328-bp partial cDNA sequence with high homology
(~62% 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 polypeptide of 366 amino acids, and followed by a
471-bp 3'-untranslated 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 GenBankTM 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-
.

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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. GenBankTM
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- , GenBankTM accession
number X79483), murine and human p38- (mp38- and hp38- ,
GenBankTM accession numbers D83073 and U66243,
respectively), and human p38- (hp38- , GenBankTM
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.
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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 3'-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 (~3 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
35S-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 35S-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.

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Fig. 2.
Expression pattern of murine
p38- mRNA. Poly(A)+ RNAs
from the indicated mouse adult tissues were prepared for Northern blot
analysis and probed with the murine p38- cDNA (upper
panel). As a control, the same blot was re-probed with a -actin
cDNA to check the integrity of the RNA (bottom
panel).
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Fig. 3.
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 × 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 × magnification). C, ISH of a frontal section of a 12.5-day
embryo (2.5 × 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 × 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 × 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 × magnification).
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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 other positive locus detectable under the
condition used; therefore, the gene of p38-
was mapped to mouse
chromosome 17, region A3-B (Fig. 4C).

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Fig. 4.
Chromosomal mapping of mouse
p38- gene by fluorescence in situ
hybridization (FISH). A, FISH signals of mouse
p38- on a representative metaphase spread. B, the
respective DAPI banding patterns of the mouse chromosomes.
C, schematic representation of map assignments for several
metaphase spreads. Each dot represents one metaphase spread
that showed a signal at the indicated mouse chromosome band.
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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).

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Fig. 5.
Chromosomal mapping of human
p38- gene by fluorescence in situ
hybridization (FISH). A, FISH signals of human
p38- on a representative metaphase spread. B, the
respective DAPI banding patterns of the human chromosomes.
C, schematic representation of map assignments for several
metaphase spreads. Each dot represents one metaphase spread
that showed a signal at the indicated human chromosome band.
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P38-
and P38-
Differ in Substrate Specificity--
To
investigate whether p38-
and p38-
share substrate specificity, we
transfected either the Flag-tagged human p38-
or the Flag-tagged
murine p38-
cDNA into 293T cells and prepared lysates from
transfected cells in the presence of H2O2
stimulation. After immunoprecipitation with anti-Flag M2 mAb, the
p38-
or p38-
kinase activity was determined by an immunocomplex
kinase assay, using myelin basic protein (MBP), GST-c-Jun, GST-MAPKAP
kinase-2, PHAS-1, and ATF-2-(1-96) as substrates with or without
p38-
inhibitor. The immunocomplex kinase assay detected marked
phosphorylation of PHAS-1 and ATF-2 by p38-
but little
phosphorylation of PHAS-1 and ATF-2 in the presence of p38-
inhibitor (Fig. 6A). In
contrast, p38-
phosphorylated PHAS-1 and ATF-2 strongly in the
absence or presence of p38-
inhibitor (Fig. 6B). Although
p38-
phosphorylated MAPKAP kinase-2 significantly but not GST-c-Jun
(Fig. 6A), p38-
phosphorylated GST-c-Jun but not MAPKAP
kinase-2 (Fig. 6B). As positive controls, both p38-
and
p38-
phosphorylated MBP (Fig. 6, A and B). As
a negative control, an empty vector alone was transfected into 293T
cells, and no detectable p38-
or p38-
kinase activity was found
(data not shown).

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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 µg each). pVA1 plasmid (10 µg) 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.
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To determine whether serine and threonine were major sites of substrate
phosphorylation by p38-
and p38-
, we isolated the 32P-labeled band of ATF-2-(1-96) from the polyacrylamide
gel of the immunocomplex kinase assays as described above (Fig. 6,
A and B) and performed phosphoamino acid analysis
(49). Phosphoamino acid analysis of the p38-
- and
p38-
-phosphorylated ATF-2-(1-96) detected Thr(P) and/or Ser(P) only
(Fig. 6, C and D), indicating that p38-
and
p38-
are indeed serine/threonine kinases.
P38-
Is Activated by Cellular Stress, Proinflammatory Cytokines,
and MAPK Kinases (MKKs)--
Because members of the p38 MAPK family
are activated by environmental stresses, proinflammatory cytokines, and
MAPK kinases (MKK-3 or MKK-4, or MKK-6) (7, 8, 17, 44), we investigated whether these stimulants and upstream kinases could induce the p38-
kinase activity also. 293T cells were transfected with the Flag-tagged
murine p38-
cDNA and treated with a variety of stimulants or
proinflammatory cytokines. In addition, 293T cells were co-transfected with p38-
cDNA plus an empty vector or MKK-3 or MKK-4 or MKK-6 or MKK-7 or MEK kinase-5 (ASK-1) cDNA. After immunoprecipitation with anti-Flag M2 mAb, the p38-
kinase activity was determined by an
immunocomplex kinase assay, using ATF-2-(1-96) as a substrate. As
expected, p38-
was activated significantly (approximately 3-5-fold)
by the various stimuli tested and proinflammatory cytokines TNF-
,
IL-1
, and epidermal growth factor (EGF) (Fig.
7A). Furthermore, co-transfection of 293T cells with p38-
plus MKK-3 or MKK-4 or MKK-6
or MKK-7 resulted in strong activation of the p38-
kinase activity,
whereas co-transfection of cells with p38-
and ASK-1 showed no
detectable effect (Fig. 7B). As a control for p38-
protein expression, equal amounts of each cell lysate were resolved by
SDS-PAGE and immunoblotted with anti-Flag M2 mAb. The data showed
comparable levels of p38-
protein expression in the corresponding samples (Fig. 7, bottom panel). Thus, p38-
is activated
by environmental stress, proinflammatory cytokines, and upstream
kinases (MKK-3, -4, -6, and -7).

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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 µg each) in the absence or
presence of extracellular stimuli as indicated. pVA1 plasmid (10 µg)
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 µg) were resolved by 10% SDS-PAGE and immunoblotted with
anti-Flag M2 mAb (bottom panel). B, the
Flag-tagged murine p38- cDNA was transfected into 293T cells
with either an empty vector (negative control, lane 1) or
the indicated MAPK kinase (MKK) expression plasmids (lanes
2-6) or MEK kinase-5 (ASK-1). As negative controls, 293T cells
were either co-transfected with the Flag-tagged mutant p38- (AGF)
cDNA plus MKK-3 cDNA (lane 7) or MKK-6 cDNA
(lane 8) or transfected with the p38- (AGF) cDNA alone
in the presence of H2O2 stimulation (lane
9).
|
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The Dual Phosphorylation TGY Motif Is Essential for Activation of
P38-
--
Since activation of p38 MAPK is mediated by dual
phosphorylation on Thr180 and Tyr182 within the
kinase domain (17), we investigated whether this Thr180-Gly-Tyr182 motif was essential for
p38-
activation. A mutant p38-
was constructed by substituting
the Thr180-Gly-Tyr182 motif with
Ala180-Gly-Phe182 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 H2O2
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 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 Thr180-X-Tyr182 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 Thr180 and
Tyr182 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 Thr180 and
Tyr182. P38-
is also activated by its upstream kinases
(MKKs) which may phosphorylate Thr180 and/or
Tyr182 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 activated 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 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.