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INTRODUCTION |
The p53 tumor suppressor protein is a transcription factor that
enhances the transcriptional rate of several known genes, which play a
critical role in transducing a signal from damaged DNA to specific
cellular response (1-5). Previous studies have demonstrated that p53
protein contains four major functional domains (6, 7). At the N
terminus is a transcriptional activation domain (amino acids 1-43) and
within the central part of p53 is the sequence-specific DNA-binding
domain (amino acids 100-300) (8). The C-terminal portion contains an
oligomerization domain and a regulatory domain (amino acids 319-393)
(9). These domains of p53 were defined in terms of separable activities
that contribute to its overall functions. A transcriptional activation
domain has been found for several regulatory proteins, such as GADD45 (10), mdm (11), WAF1/p21/CIP1 (12), and cyclin
G (13). The DNA-binding domain accommodates most of the mutations found so far (6), and a regulatory domain has been implicated in binding to
damaged DNA (14) and in apoptosis (15).
The activation of p53 has been implicated in cell cycle control, DNA
repair, and apoptosis (1, 2, 16-18). Its function is controlled at the
levels of transcription, translation, protein turnover, and cellular
compartmentalization, as well as association with other proteins (19).
In addition to these conditions, growing evidence indicates that the
ability of p53 to inhibit diverse regulatory functions is also likely
to depend on its phosphorylation, which is
conformation-dependent (7, 20, 21). There are at least
seven phosphorylation sites within the N terminus of the p53 protein
and several phosphorylation sites in the C terminus (19). It was
reported that some of these sites may influence DNA binding (20, 22),
transactivation (20, 23), and growth arrest (24). p53 phosphorylation
is mediated by a variety of protein kinases, including casein kinase
(CK)1 I, CK II, protein
kinase A, CDK7, DNA-activated protein kinase, JNKs, ERKs, or protein
kinase C (7, 19, 21, 24-27). It was found that phosphorylation of
murine p53 protein at serine 389 (or homolog serine 392 of the human
p53 protein) caused the enhancement of p53 DNA binding activity
in vitro (28). Very recently, two different research groups
reported that UV radiation induced the phosphorylation of p53 at serine
389 and that this phosphorylation is important for p53-mediated
transcriptional activation in vivo (29, 30). Furthermore,
Kapoor et al. (30) found that CKII could phosphorylate the
p53 at serine 389 in vitro. Here, we demonstrated that
UVC-induced p53 phosphorylation at serine 389 is mediated by p38 kinase
by using a dominant negative mutant of p38 kinase and the p38 kinase
inhibitor, SB202190, both in vitro and in
vivo.
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EXPERIMENTAL PROCEDURES |
Materials--
Activated p38 kinase and active JNK2 were from
Upstate Biotechnology; activated ERK2, Elk-1 fusion protein, c-Jun
fusion protein, phospho-specific c-Jun (Ser63) antibody,
phospho-specific Elk-1 antibody (Ser383), p38 kinase
antibody, phospho-specific p38 kinase (Tyr182) antibody,
phospho-specific p53 (Ser389) antibody, and p38 kinase
assay kit were purchased from New England Biolabs; monoclonal mouse IgG
against p53 antibody (Ab1) was from Oncogene Research Products; MEK1
specific inhibitor, PD98059, was from Biomol; p38 kinase inhibitor,
SB202190, was from Calbiochem; dominant negative mutant of p38 kinase
was a generous gift from Dr. Mercedes Rincon, Department of Medicine,
University of Vermont, Burlington, VT (31, 32); Eagle's minimal
essential medium (MEM), Dulbecco's modified Eagle's medium, and RPMI
1640 were from Calbiochem; fetal bovine serum (FBS) was from Life
Technologies, Inc.; and luciferase substrate was from Promega.
Recombinant p53 was produced in insect cells infected with baculovirus
vector carrying human p53 cDNA and partially purified through DNA
affinity chromatography (33).
Cell Culture--
JB6 mouse epidermal cell line Cl 41 and its
stable transfectants, Cl 41 CMV-neu, Cl 41 DN-p38 G7, Cl 41 DN-JNK1
mass1, Cl 41 MAPK-DN B3 mass1, and
Cl 41 p53 were cultured in monolayers at 37 °C and 5%
CO2 using Eagle's MEM containing 5% FBS, 2 mM L-glutamine, 25 µg/ml gentamicin (18, 34, 35).
Immunoprecipitation Assay--
The level of p53 phosphorylation
at serine 389 induced by UVC radiation and the complex of p53 with p38
kinase was measured by Western blot for immunoprecipitation using
specific antibodies against p53. Briefly, JB6 Cl 41 cells or its
transfectants were cultured in 100-mm dishes with 5% FBS MEM until
they reached 80% confluence. Then, the cells were starved by culturing
them in 0.1% FBS MEM for 48 h. The cells were exposed to UVC
irradiation for induction of p53 phosphorylation at serine 389. The
cells were lysed on ice for 1 h in the lysis buffer and spun at
14,000 rpm for 5 min. The lysates were immunoprecipitated using p53
antibodies (Ab1) and protein G plus protein A-agarose. The bands were
washed, and the phosphorylated protein of p53 at serine 389 and the p38 kinase, as well as phosphorylated p38 kinase, were selectively measured
by Western immunoblotting using a specific antibody and chemiluminescent detection system.
Generation of Stable Cotransfectants--
JB6 Cl 41 cells were
cultured in a six-well plate until they reached 85-90% confluence. We
used 1 µg of CMV-neo vector with or without 12 µg of plasmid DNA of
dominant negative mutant of p38 kinase and 15 µl of LipofectAMINE
reagent to transfect each well in the absence of serum. After 10-12 h,
the medium was replaced by 5% FBS MEM. Approximately 30-36 h after
the beginning of the transfection, the cells were digested with 0.033%
trypsin, and cell suspensions were plated into 75-ml culture flasks and
cultured for 24-28 days with G418 selection (300 µg/ml). Stable
transfectants were identified by using p38 kinase activity assay kit.
Stable transfected Cl 41 CMV-neu and Cl 41 DN-p38 G7 were established and cultured in G418-free MEM for at least two passages before each experiment.
Assay for p38 Kinase Activity--
p38 kinase assay was carried
out as described by the protocol of New England Biolabs. In brief, JB6
Cl 41 CMV-neu or Cl 40 DN-p38 G7 cells were starved for 48 h in
0.1% FBS MEM at 37 °C, 5% CO2 atmosphere incubator.
The cells were washed once with ice-cold phosphate-buffered saline and
were exposed to UVC (50 KJ/m2) for 30 min. Then, the cells
were washed once with ice-cold phosphate-buffered saline and lysed in
300 µl of lysis buffer per sample (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
-glycerolphosphate, 1 mM Na3VO4,
1 mg/ml leupeptin). The lysates were sonicated and centrifuged and the supernatant incubated with a specific p38 kinase antibody with gentle
rocking for 4-10 h at 4 °C and then the protein A-Sepharose beads
were added in the incubation for another 4 h. The beads were
washed twice with 500 ml of lysis buffer with phenylmethylsulfonyl fluoride and twice with 500 µl of kinase buffer (25 mM
Tris, pH 7.5, 5 mM
-glycerolphosphate, 2 mM
DTT, 0.1 mM Na3VO4, 10 mM MgCl2). The kinase reactions were carried
out in the presence of 100 µM ATP and 2 µg of ATF-2 at
30 °C for 30 min. ATF-2 phosphorylation is selectively measured by
Western immunoblotting using a chemiluminescent detection system and
specific antibodies against phosphorylation of ATF-2 at
Thr71.
Protein Phosphorylation Assay in Vitro--
Phosphorylation of
p53, c-Jun, or Elk-1 by activated ERK2, JNK2, or p38 kinase were
carried out at 30 °C for 60 min in the presence of kinase buffer (25 mM Tris, pH 7.5, 5 mM
-glycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4,
10 mm MgCl2) with 200 µM ATP and p53, c-Jun,
or Elk-1 as substrate. The phosphorylation proteins were detected by
Western immunoblotting using phospho-specific antibodies.
Gel Mobility Shift Assay--
The p53 DNA-protein binding assay
was carried out according to methods described previously (37).
The synthetic oligonucleotide of p53-binding consensus sequence
(WAF-1: 5'-GAACATGTCCCAACATGTTG-3' or GADD45:
5'-GAACATGTCTAAGCATGCTG-3') was annealed and labeled with
32P using T4 polynucleotide kinase and
[
-32P]ATP. Nuclear extracts were prepared from
confluent Cl 41 cells treated with various concentrations of SB202190
for 1 h, then exposed to UVC (60 J/m2) and cultured
for 18 h. Cells were washed with phosphate-buffered saline and
lysed in cold lysis buffer (25 mM HEPES, pH 7.8, 50 mM KCl, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml
aprotinin, and 100 µM DTT) for 5 min. After washing once
with the lysis buffer without Nonidet P-40, the pellet was resuspended
in cold extraction buffer (25 mM HEPES, pH 7.8, 500 mM KCl, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml
aprotinin, and 100 µM DTT) and incubated on ice for 20 min with frequent mixing followed by spinning at 14,000 rpm for 5 min.
The supernatant was then saved as the nuclear extract. Protein
concentrations were determined using the Bio-Rad Protein Assay Kit. A
DNA-binding reaction mixture of 24 µl contained 20 mM
Tris-HCl, pH 7.5, 4% Ficoll-400, 2 mM EDTA, 0.5 mM DTT, 1 µg of poly(dI/dC), 32P-labeled
oligonucleotide (20,000 cpm) was incubated with 6 µg of
protein-containing nuclear extract. To determine the binding specificity, 100 × excess of cold oligonucleotide was included in
some reactions. The mixture was incubated at room temperature for 45 min and then loaded onto a 3.5% polyacrylamide gel. The gel was run in
0.5 × Tris borate-EDTA buffer at 160 V, dried, and scanned.
Assay for p53-dependent Transcription
Activity--
p53-dependent transcription activity was
assayed by using a Cl 41 cell line stably expressing a luciferase
reporter gene, which was controlled by p53 DNA binding sequences (18,
38). Confluent monolayers of Cl 41 p53 cells (18) were trypsinized and
8 × 103 viable cells suspended in 100 µl of 5% FBS
MEM were added into each well of a 96-well plate. Plates were incubated
at 37 °C in a humidified atmosphere of 5% CO2. Twelve
to 24 h later, cells were starved by culturing them in 0.1% FBS
MEM for 12 h. The cells were treated with different concentrations
of SB202190 for 1 h, then exposed to UVC (60 J/m2) for
p53 induction and cultured for 24 h. The cells were extracted with
lysis buffer, and luciferase activity was measured using a luminometer
(Monolight 2010). The results are expressed as relative p53 activity
(18).
 |
RESULTS |
Induction of p53 Phosphorylation at Serine 389 by UVC
Radiation--
P53 activity is regulated by multisite phosphorylation
(7, 20, 21, 29, 30). It has been reported that phosphorylation of the
murine p53 protein at serine 389 in UV response plays an important role
in p53 sequence-specific DNA binding in vitro and in
p53-dependent transcriptional activation in vivo
(28-30). To investigate the signal transduction pathways leading to
phosphorylation of p53 at serine 389 in UV response, we first measured
the UVC-induced phosphorylation of p53 at serine 389 in a mouse JB6
epidermal cell line, Cl 41. Previous studies indicated that Cl 41 cells contain high levels of wild-type p53 protein that could activate the
p53-dependent transcription in UV response (18, 36-38). Cl 41 cells were exposed to UVC radiation; the p53 protein in the cell
lysate was immunoprecipitated with specific antibodies against p53
(Ab1). The levels of p53 phosphorylation were analyzed by Western
blotting using phospho-specific antibody against p53 at serine 389. The
results show that UVC radiation caused phosphorylation of p53 at serine
389 (Fig. 1). This phosphorylation was
observed at 30 and 90 min after UVC exposure (Fig. 1A). The
dose response study indicated that 60 J/m2 UVC is the
optimal dosage for induction of p53 phosphorylation at serine 389 (Fig.
1B). These results are in agreement with previous findings
that UV radiation leads to increased phosphorylation of p53 at serine
389 (29, 30).

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Fig. 1.
Induction of p53 phosphorylation by UVC
radiation. Cl 41 cells were cultured in monolayers in 100-mm
diameter dishes until 90% confluent. The cells were starved by
changing the medium with 0.1% FBS MEM for 24-48 h. Then, the cells
were exposed to UVC (60 J/m2) for different time
incubations (A) or different doses of UVC for 30 min
(B). The cells were harvested, and the phosphorylation
levels of p53 at serine 389 were measured as described under
"Experimental Procedures."
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Inhibition of UVC-induced p53 Phosphorylation at Serine 389 by
Pretreatment of Cells with SB202190, but Not with PD98059--
UV
radiation leads to activation of MAP kinase superfamily, comprising
ERKs, JNKs, and p38 kinase (39-41). Previous studies have demonstrated
that ERKs and JNKs are responsible for phosphorylation of p53 at
threonines 73 and 83 or serine 34, respectively (7, 21, 24, 26, 27).
Since the UV radiation also leads to increased phosphorylation of p53
at serine 389, we investigated the possible role of MAP kinase family
in this process. To test this possibility, we first examined the
influence of specific chemical inhibitors on UVC-induced
phosphorylation of p53 at serine 389. The results are shown in Fig.
2. UVC-induced phosphorylation of p53 at
serine 389 was completely impaired by pretreatment of cells with 1-2
µM SB202190, a specific inhibitor for p38 kinase, while
25-100 µM PD98059, which showed marked inhibition of
UV-induced activation of MEK1 and ERKs (data not shown), did not show
any inhibition of p53 phosphorylation at serine 389 (Fig. 2). These data suggest that p38 kinase may be involved in p53 phosphorylation at
serine 389 in UVC response.

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Fig. 2.
Inhibition of UVC-induced p53 phosphorylation
at serine 389 by pretreatment of cells with p38 kinase inhibitor,
SB202190, but not with MEK1 inhibitor, PD98059. Cl 41 cells were
cultured in monolayers in 100-mm diameter dishes until 90% confluent.
The cells were starved by changing the medium with 0.1% FBS MEM for
36-48 h. Then, the cells were pretreated with either SB202190
(A) or PD98059 (B) for 1 h at the
concentration indicated. The cells were exposed to UVC (60 J/m2) for 30-min incubation. Thirty minutes later, the
cells were harvested, and phosphorylation levels of p53 at serine-389
were measured as described under "Experimental Procedures."
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Blocking p53 Phosphorylation at Serine 389 by Expressing a Dominant
Negative Mutant of p38 Kinase, but Not by Dominant Negative Mutants of
ERK2 or JNK1--
To directly investigate the role of p38 kinase in
UVC-induced phosphorylation of p53 at serine 389, we established a
stable transfectant with a dominant negative mutant of p38 kinase (31, 32). The dominant negative mutant of p38 kinase was generated by
replacing Thr180 and Tyr182 by Ala and Phe,
respectively (31, 32). Cl 41 cells were transfected with dominant
negative mutant of p38 kinase and selected with G418 as described under
"Experimental Procedures." G418-resistant transfectants were
analyzed for the expression of dominant negative mutant by assay for
UVC-induced p38 kinase activity. The results showed that expression of
dominant negative mutant of p38 kinase specifically blocked UVC-induced
p38 kinase activity (Fig. 3), while it
did not inhibit the activation of ERKs and JNKs (data not shown). The
expression of dominant negative mutant of p38 kinase markedly blocked
p53 phosphorylation at serine 389 in UVC response (Fig.
4A). In contrast,
overexpression of dominant negative ERK2 or JNK1 did not show
significant inhibition of p53 phosphorylation at serine 389 (Fig. 4,
B and C). The time course and dose response studies further indicated that p38 kinase appears to be the mediator of
p53 phosphorylation at serine 389 in UVC response (Fig.
5).

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Fig. 3.
Expression of dominant negative p38 kinase
blocks the activation of p38 kinase induced by UVC radiation. Cl
41 cell stable transfectants, Cl 41 CMV-neu or Cl 41 DN-p38 G7, were
seeded in 100-mm dishes and cultured until 90% confluent. The cells
were starved by changing to 0.1% FBS MEM medium for 36-48 h. Then,
the cells were exposed to UVC (60 J/m2) for 30 min. The
cells were harvested, and the p38 kinase activity was determined by
protocol of New England Biolabs.
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Fig. 4.
Blocking p53 phosphorylation at serine 389 by
dominant negative mutant of p38 kinase. Cl 41 cell stable
transfectants (as indicated) were seeded in 100-mm dishes and cultured
until 90% confluent. The cells were starved by changing the 0.1% FBS
MEM for 36-48 h. The cells were exposed to UVC (60 J/m2)
for 30 min. Then, the cells were harvested, and phosphorylation levels
of p53 at serine 389 were detected as described under "Experimental
Procedures."
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Fig. 5.
Time course and dose response studies of
blocking activity by dominant negative mutant of p38 kinase on
UVC-induced p53 phosphorylation at serine 389. Cl 41 cell stable
transfectants (as indicated) were seeded and cultured in 100-mm dishes
until 90% confluent. The cells were starved by changing to 0.1% FBS
MEM for 36-48 h. Then, the cells were exposed to UVC at the dosage
indicated and cultured for 30 min (A) or UVC (60 J/m2) (B) and cultured for the time period
indicated. The cells were harvested, and phosphorylation levels of p53
were measured as described under "Experimental Procedures."
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p38 Kinase Was Present in the Immunoprecipitation Proteins from Cl
41 Cells Lysate with p53-specific Monoclonal Antibodies--
Because
the above data revealed the important role of p38 kinase in the
signaling pathway leading to phosphorylation of p53 at serine 389, we
explored whether a direct interaction might exist between p38 kinase
and p53. We exposed the Cl 41 cells to UVC radiation for the different
times and incubated the cell extract with specific monoclonal
antibodies against p53 and protein G plus protein A-agarose. The beads
were washed extensively to eliminate nonspecific binding. The
bead-coupled proteins were eluted with SDS sample buffer and measured
by Western blot using specific antibodies against phosphorylation of
p53 at serine 389 and phosphorylation of p38 kinase at threonine 182 or
nonphosphorylated p38 kinase. We found that the p38 kinase binding to
p53 was detected in the immunoprecipitation complex (Fig.
6). Furthermore, the kinetics of p53
phosphorylation at serine 389 were well correlated with that of p38
kinase phosphorylation at threonine 182 after UVC exposure (Fig. 6).
Very interestingly, the total p38 kinase in all the immunoprecipitation
samples was almost the same (Fig. 6). These data suggested that
nonphosphorylated p38 kinase could bind to p53, and the phosphorylated
p38 kinase may be responsible for p53 phosphorylation.

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Fig. 6.
In vivo association of p38 kinase
with p53. Cl 41 cells were seeded and cultured in 100-mm dishes
until 90% confluent. The cells were starved for 36-48 h in 0.1% FBS
MEM. Then, the cells were exposed to UVC (60 J/m2) for the
time period indicated. The cells were harvested, and the cell extract
was incubated with specific monoclonal antibodies against p53 and
protein G plus protein A-agarose. The beads were washed extensively,
and the bead-coupled proteins were eluted with SDS sample buffer and
measured by Western blot using specific antibodies against
phosphorylation of p53 at serine 389 and phosphorylation of p38 kinase
at threonine 182 or nonphosphorylated p38 kinase.
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P53 Is Phosphorylated at Serine 389 in Vitro by Active p38 Kinase,
but Not by Active ERK2 or JNK2--
The above data strongly suggest
that p38 kinase is the possible direct mediator of UVC-induced p53
phosphorylation at serine 389. If this is the case, the active p38
kinase should phosphorylate p53 at serine 389 in vitro. To
test this, we incubated partially purified p53 protein with one of
activated MAP kinase family in the presence of 200 µM
ATP. The phosphorylation level of p53 was detected by Western blot
using specific antibodies. The results are shown in Fig.
7. The p53 proteins were phosphorylated
by active p38 kinase (Fig. 7A), but not by active ERK2 or
JNK2 (Fig. 7, B and C), while active ERK2 and
JNK2 show their ability to phosphorylate Elk-1 or c-Jun, respectively
(Fig. 7, B and C). Because the p53 used in this
study consists of the purified baculovirus-expressed proteins that are
a mixture of phosphorylated and unphosphorylated p53 at serine 389, a
relatively high basal level of phosphorylation of p53 at serine 389 in
the control group is probably due to a mixture of phosphorylated and
unphosphorylated p53 at serine 389 in this baculovirus-expressed
protein. These data, taken together with other results from this study,
strongly suggest that p38 kinase is the direct mediator of UV-induced
phosphorylation of p53 at serine 389.

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Fig. 7.
p53 is phosphorylated at serine 389 in
vitro by active p38 kinase, but not by active ERK2 or
JNK2. Phosphorylation of p53 at serine 389 (A-C) or
the phosphorylation of c-Jun at serine 63 and Elk1 at
serine 389 was carried out at 30 °C for 60 min in the presence of
specific substrate, kinase buffer, 200 µM ATP, and one of
the MAP kinase family. The phosphorylation proteins were detected by
Western blot using specific phosphorylation antibodies.
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Blockade of p53-dependent Transcription Activity and
p53 DNA Binding Activity by Pretreatment of Cells with
SB202190--
To investigate the functional requirement of p38 kinase
mediating the phosphorylation of p53 at serine 389 in UV response, we
determined the inhibitory effect of SB202190, a p38 kinase inhibitor,
on p53-dependent transcription activity and DNA binding activity. The results showed that UVC-induced p53-dependent
transcription activity and DNA binding activity could be blocked by
pretreatment of cells with SB202190 (Fig.
8). This band was shown to be compatible by the unlabeled p53 binding oligonucleotides and was not detectable in
p53
/
cells derived from p53 knockout mouse (Fig. 8, A
and B, and data not shown). This result suggests that
phosphorylation of p53 at serine 389 is functionally required for
p53-dependent transcription.

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Fig. 8.
Pretreatment of cells with SB202190 impaired
the UV-induced p53-dependent transcription and DNA binding
activity. A and B, for DNA binding assay,
JB6 Cl 41 cells suspended in 5% FBS MEM were seeded into 100-mm dishes
and cultured until 80-90% confluent. Cells were starved in 0.1% FBS
MEM for 48 h. The cells were first treated with different
concentrations of SB202190, then exposed to UVC radiation (60 J/m2). After culturing, the cells were radiated for 18 h, then they were harvested. Nuclear extract preparation and p53 DNA
binding gel shift assay were carried out according to the method
described under "Experimental Procedures." The synthetic
oligonucleotide sequences are: WAF-1, 5'GAACATGTCCCAACATGTTG-3'
(A) and GADD45, 5'-GAACATGTCTAAGCATGCTG-3' (B).
C, for p53-dependent transcription assay, Cl 41 p53 cells were seeded in 96-well plates and cultured until 80-90%
confluent. The cells were starved in 0.1% FBS MEM for 12 h. Then
the cells were treated with SB202190 for 1 h at the concentration
indicated. The cells were exposed to UVC (60 J/m2) and
cultured for 24 h. The luciferase activity was measured, and the
results were expressed as relative p53 activity (18).
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|
 |
DISCUSSION |
DNA damage-induced activation of the p53 tumor suppressor is
suggested to be central in cellular damage response pathways. One of
the major roles of p53 in normal cells is to trigger cell cycle arrest
or apoptosis in response to DNA damage by acting as a sequence-specific
transcription factor that activated genes involved in control of cell
cycle and apoptosis (20). Mutations in the p53 gene have been found in
over half of all human cancers (20). Loss of p53 suppressor function,
by mutation, is a universal step in the development of human cancer
(42). For these reasons, it was very interesting to investigate the
signal transduction pathways leading to p53 phosphorylation and
activation. UV radiation has been shown previously to induce p53
phosphorylation at serine 389. The critical kinase that mediates this
UV-responsive phosphorylation is, however, not well documented. In this
study, we determined the role of p38 kinase in UVC-induced p53
phosphorylation at serine-389. Exposure of Cl 41 cells to UVC radiation
leads to activation of p38 kinase and the phosphorylation of p53 at
serine 389 in Cl 41 cells. Pretreatment of cells with SB202190, a p38
kinase inhibitor, or expressing of dominant negative mutant of p38
kinase, impaired the phosphorylation of p53 at serine 389 and
p53-dependent transactivation and DNA binding activity in
UVC response. Most importantly, we found that p38 kinase was present in
the immunoprecipitation proteins from a cell extract using specific
antibodies against p53, and active p38 kinase was shown to
phosphorylate the p53 protein at serine 389 in vitro. All
these data demonstrated that p38 kinase plays a critical role in
UVC-induced phosphorylation of p53 protein at serine 389, suggesting
that there is a functional requirement of p38 kinase for UV-induced p53 activation.
Exposure of cells to UV irradiation elicits a complex set of acute
cellular responses called "UV responses." Generally, UV responses
serve to protect the cells. The initial signal triggering the UV
response is in large part independent of DNA damage, but rather appears
to be mediated by a membrane-associated component of the Ras pathway
with activation of MAPKs (39, 40, 43, 44). However, others argue that
even UV-induced activation of MAPKs may have a DNA damage signal
component (45). Very recently, Bender et al. (46) reported
that UV-induced activation of NF
B is through DNA
damage-dependent and -independent pathways. In the case of
p53, activating signals clearly involve both DNA damage signals and
other nongenotoxic stresses (47). It is accepted that p53 function is
regulated at the levels of its transcription, translocation, protein
turnover, cellular compartmentalization, stabilization, and association
with other proteins, as well as phosphorylation (19). p53 has multiple
sites for phosphorylation in both N- and C-terminal domains, and p53
phosphorylation has been found to be mediated by several cellular
kinases, including CKI, CKII, PKA, CDK7, ERKs, and JNKs (7, 19, 21,
24-27). Among these protein kinases, those that are expected to
produce stress-activated phosphorylation of p53 are JNKs and
DNA-activated protein kinase, which reportedly phosphorylates residues
of p53 within the N-terminal domain of p53 (19, 21). DNA-activated protein kinase mediates phosphorylation of p53 at serine 15 and serine
37 (48). This phosphorylation was identified as one of the major sites
on p53 in cellular stress response and contributed to p53 accumulation
(48). JNKs phosphorylate the murine p53 at serine 34 (19, 26, 27). JNKs
signaling contributes to the ability of p53 to mediate apoptosis
through stabilization and activation of p53 (7, 21). It was also
reported that the DNA binding function of p53 is activated by
phosphorylation of the C-terminal serine by purified CKII in
vitro (28). The studies from two different groups indicated that
UV radiation results in functional activation of p53 through
phosphorylation of p53 protein at serine 389 (29, 30) and that p53
could be phosphorylated at serine 389 by CKII in vitro (30).
In this study, we found that inhibition of p38 kinase by either
chemical p38 kinase inhibitor SB202190, or the biological inhibitor,
dominant negative mutant of p38 kinase, results in blocking
phosphorylation of p53 at serine 389 in UV radiation. Pretreatment of
cells with SB202190 also impaired the UV-induced
p53-dependent transcription and DNA binding activity. Most
importantly, the p38 kinase not only phosphorylates the p53 at serine
389 both in vitro and in vivo, but also is
present in the immunoprecipitation proteins from cell lysate using
specific antibodies against p53. Furthermore, the phosphorylation at
serine 389 is well correlated with the phosphorylation of p38 kinase,
which is considered essential for activation of p38 kinase, in the time
course studied. Taken together, our results provide evidence, both
in vitro and in vivo, that p38 kinase is a
mediator of p53 phosphorylation at serine 389 in UVC response, and p38
kinase is functionally required for UV-induced p53-dependent transcription.