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Molecular and Cellular Biology, April 2001, p. 2743-2754, Vol. 21, No. 8
The Ruttenberg Cancer
Center1 and Department of Cell
Biology,4 Mount Sinai School of Medicine, New
York, Department of Cell Biology and Anatomy, New York Medical
College, Valhalla,5 and Department of
Pathology and Laboratory Medicine, State University of New York Health
Science Center, Brooklyn,7 New York;
Cell Stress and Aging Section, Laboratory of Biological
Chemistry, National Institute on Aging, National Institutes of Health,
Baltimore, Maryland2; Department of
Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel
Aviv University, Tel Aviv, Israel3;
Cancer Research Institute, Kanazawa University, Kanazawa,
Japan6; and Isis Pharmaceuticals Inc.,
Carlsbad, California8
Received 9 October 2000/Returned for modification 28 November
2000/Accepted 24 January 2001
The p53 tumor suppressor protein plays a key role in the regulation
of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and
transcriptional activities. Mass spectrometry analysis of p53
phosphorylated in 293T cells by active Jun NH2-terminal
kinase (JNK) identified T81 as the JNK phosphorylation site. JNK
phosphorylated p53 at T81 in response to DNA damage and stress-inducing
agents, as determined by phospho-specific antibodies to T81. Unlike
wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did
not exhibit increased expression or concomitant activation of
transcriptional activity, growth inhibition, and apoptosis. Forced
expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells
decreased T81 phosphorylation while reducing p53 transcriptional
activity and p53-mediated apoptosis. Similarly transfection of
antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV
irradiation. More than 180 human tumors have been reported to contain
p53 with mutations within the region that encompasses T81 and the JNK
binding site (amino acids 81 to 116). Our studies identify an
additional mechanism for the regulation of p53 stability and functional
activities in response to stress.
The TP53 gene encodes a
short-lived transcription factor implicated in the regulation of cell
cycle progression, DNA repair, replicative senescence, and programmed
cell death (21, 43, 47). The p53 protein is normally
expressed at low levels in nonstressed normal growing cells. In
response to DNA damage or stress, p53 is modified posttranslationally,
with resultant accumulation of transcriptionally active protein
(21, 43, 47). Activation of p53 in response to stress
leads to temporary growth arrest or apoptosis. Mutations in
p53, which are common in human tumors, abrogate its ability
to contribute either to growth arrest or to cell death (43,
60). Most mutations found within the DNA-binding domain of p53
impair its binding to target DNA sequences (58). Different
p53-responsive genes mediate a variety of cellular functions, including
growth arrest by p21WAF1/CIP1 and 14-3-3 (12,
28) and apoptosis by Bax, CD95/Fas, IGF-BP3, and p53-inducible
genes (6, 39, 44, 45). However, the mechanisms that enable
activation of a selective subset of p53 target genes are poorly understood.
Accumulating evidence suggests that the regulation of p53's
transcriptional activities depends on the nature of its phosphorylation (3, 16, 21, 37). Furthermore, a key factor that determines p53's ability to mediate its multiple activities is its stability, which is tightly regulated by Mdm2 and Jun NH2-terminal
kinase (JNK) in a phosphorylation-dependent manner (8, 17, 18, 21, 25, 34). In nonstressed cells, Mdm2 targeting of p53 ubiquitination primarily occurs in the G2/M phase of the
cell cycle, whereas JNK association and targeting of p53 for
ubiquitination take place during the G0 phase
(17). In response to stress, p53 phosphorylation coincides
with its dissociation from both Mdm2 and JNK (17, 51).
More than a dozen phosphorylation sites have been mapped on p53, but
only a few have been characterized with respect to the phosphorylating
kinase and the relevance of the phosphorylation to p53 activities.
Within the C terminus, human p53 is phosphorylated at amino acids 315, 378, 389, and 392, which enhances the in vitro specific DNA-binding
activity of p53 (30, 31, 36, 54). At the p53 N terminus,
several potential phosphorylation sites have been identified, including
Ser 6, 9, 15, 20, 33, 37, and 46 and Thr 18 and 55 (11, 20, 21,
32, 37). ATM (ataxia telangiectasia mutated) has been implicated
in Ser 15 phosphorylation (4, 40, 51, 53), which is
induced by DNA damage (40, 53) and which is associated
with enhanced p53 transcriptional activities (11, 49, 51).
Gamma irradiation-induced ATM-dependent activation of Chk2 results in
phosphorylation of Ser 20, which has been implicated in p53
transcriptional activities (10, 29), p53 tetramerization,
down-regulation of Mdm2 suppression, p53 stabilization in response to
stress (10, 57, 51, 52), and p53-mediated apoptosis
(52, 59).
Phosphorylation by p38 kinase on Ser 33 and 46 has been shown to be
required for UV-induced p53-mediated apoptosis (7), and
inhibition of p38 has been found to attenuate transcriptional activities of p53 and p53-dependent apoptosis induced by
chemotherapeutic agents (50). Phosphorylation-driven
acetylation of p53 on C-terminus sites activates sequence-specific DNA
binding (24, 49) and has also been implicated in
transcription-dependent p53-mediated apoptosis (9).
Together, these observations point to a complex array of
phosphorylation events occurring in response to stress and DNA damage that involve multiple kinases and multiple phosphoacceptor sites. The
regulation of such complex phosphorylation patterns may involve sequential phosphorylation events, as was shown for phosphorylation of
Ser 15, which depends on phosphorylation at Ser 46 (7).
In earlier studies we established the contribution of JNK to the
regulation of p53 stability. In cells in the G0 and
G1 phases of the cell cycle, among other specific
conditions, JNK but not Mdm2 is associated with p53 and JNK efficiently
targets the ubiquitination and degradation of p53; similarly, JNK
serves as a targeting molecule for ubiquitination of nonphosphorylated
c-Jun (15), ATF2 (19), and JunB
(14). The JNK association with p53 has been mapped to
residues 97 to 116 (2), whose deletion prolongs p53's
half-life (17). Expression of constitutively active JNK
kinase (JNKK) upstream kinase MEKK1 increases p53 stability and
transcriptional activity (18). Given the multiple
downstream effectors of MEKK1, we further elucidated the possible
contribution of JNK to p53's stability and activities. Here we
identify and characterize T81 as the phosphoacceptor site for JNK
phosphorylation. We show that phosphorylation of T81 in response to
stress or DNA damage stabilizes p53 and confers on it transcriptional
activities and the ability to elicit apoptosis. In the absence of JNK
expression or JNK-mediated phosphorylation, p53 is inactive. The
implications of JNK phosphorylation at T81 for p53 activities in the
context of the complex phosphorylation of p53 in response to stress are discussed.
Cells.
293T (ATCC CRL1573) human embryo kidney cells, early
passages of normal human fibroblasts (NHF; kind gift from H. Tahara), MCF7 breast cancer cells (ATCC HTB22), and p53 null cells (human lung
tumor H1299 and mouse fibroblast 10.1 cell lines; kindly provided by X. Wu, and S. Aaronson, respectively) were grown in Dulbecco modified
Eagle medium supplemented with antibiotics and 10% fetal bovine serum.
Oligonucleotide synthesis and treatment.
The
oligonucleotides used were synthesized at Isis Pharmaceuticals, Inc.
The sequences of the oligonucleotides are as follows: JNK1AS
(ISIS12439), 5'-CTC TCT GTA GGC CCG CTT GG-3'; JNK2AS
(ISIS18076), 5'-GTC CGG GCC AGG CCA AAG TC-3'; JNK1Scr
(ISIS101759), 5'-CTT TCC GTT GGA CCC CTG GG-3'; JNK2Scr
(ISIS101769), 5'-GTG CGC GCG AGC CCG AAA TC-3'. All
oligonucleotides consisted of 2'-O-methoxyethyl modified
sugars (residues 1 to 5 and 16 to 20) and 2-deoxy sugars (residues 6 to
15). The linkages are uniform phosphorothioate. Cells were treated with
0.25 µM oligonucleotides in the presence of 10 µg of Lipofectin
reagent (Life Technologies)/ml as described previously
(46).
Expression vectors.
Human p53 cDNA was used as a backbone
for generating a PCR-based site-directed single-base-pair substitution
at amino acid (aa) 81 (Quick Change; Stratagene), generating
p53T81A, which was confirmed by sequencing. Rat p53 mutated
at aa 101, 105, and 108 or with aa 97 to 116 deleted ( In vitro phosphorylation of p53 by purified JNK.
293T cells
(108) were calcium transfected with hemagglutinin
(HA)-tagged JNK2 (30 µg) and 36 h later treated with UV-C (40 J/m2). Proteins prepared within 45 to 60 min after
irradiation were subjected to immunoprecipitation using antibodies to
HA. Immunoblotting and silver staining were performed on an aliquot of
the purified material to ensure an appropriate degree of purity.
Activity of HA-JNK was verified using glutathione
S-transferase (GST)-Jun as a substrate in solid-phase
kinase reactions. Wild-type (wt) or T81A mutant forms of p53 were in
vitro translated using an in vitro translation kit (TNT; Promega). The
translated proteins were purified using p53 antibodies (pAb 421) that
were covalently bound to Amino link beads (Pierce). Bound p53 was
eluted from the antibodies using elution buffer (Pierce) followed by
dialysis against kinase buffer (20 mM HEPES [pH 7.6], 1 mM EGTA, 1 mM
dithiothreitol, 2 mM MgCl2, 2 mM MnCl2, 5 mM
NaF, 1 mM NaVO3, 50 mM NaCl). The amount and purity of p53
were verified by silver staining and immunoblotting before equal
aliquots were subjected to an in vitro solid-phase kinase reaction
(1). Briefly, bead-bound HA-JNK was incubated with p53 at
37°C for 15 min in the presence of [ Stress and DNA damage treatments.
UV irradiation was
administered as previously described (1). Briefly, cells
were subjected to UV-C irradiation (254 nm; 40 J/m2) in the
calibrated area within the tissue culture hood. Medium that had been
removed prior to irradiation was returned to minimize serum-induced
changes. Treatment with anisomycin (10 µg/ml), hydrogen peroxide (100 µM), sorbitol (0.6 mM), or cis-platinum (0.6 µM) (all
purchased from Sigma) was performed by adding them to the medium (time
zero) followed by time point analysis as indicated in Results.
Protein stability analysis.
To monitor changes in the
stability of p53, pulse-chase analysis was performed using the
previously described [35S]methionine-cysteine mixture
(17). Briefly, 10.1 p53 null cells (106) were
transfected with the wt or T81A mutant form of p53 (2 µg) using
Lipofectamine Plus reagent (GIBCO-BRL). Twenty-four hours after
transfection the cells were starved (serum without methionine) for
1 h before addition of [35S]methionine-cysteine
mixture (1 mCi) to cells for 1 h (time zero followed by chase for
the periods of time indicated by the time points in the pertinent
figures. Cells were counted and harvested, followed by protein
preparation and p53 immunoprecipitation from equal numbers of cells
(using 2 µg of DO1 antibodies per 106 cells).
Cycloheximide-chase experiments were performed on cells transfected as
indicated above, and 24 h later cycloheximide (60 µg/ml) was
added to the cultures (time zero). Equal amounts (500 µg) of proteins
prepared at various time points were subjected to immunoprecipitation
(DO1) followed by immunoblot analysis (polyclonal p53 antibody).
Mass spectrophotometry analysis.
293T cells
(108) were transfected (calcium phosphate) with p53 and
JNKK2CAA expression vectors. Twenty-four hours after
transfection, protein extracts were incubated with p53 antibodies (DO1)
immobilized on Aminolink beads (Pierce). Immunoprecipitates were eluted
from antibodies reduced and alkylated with iodoacetamide before
SDS-PAGE (22). A portion of the gel was electroblotted,
followed by immunoblotting to reveal the position of p53. Coomassie
blue-stained protein bands were excised from SDS-PAGE gels and digested
with trypsin in situ (48). The digests containing the gel
bits were extracted in 50% acetonitrile-0.1% trifluoroacetic acid
(TFA), concentrated using a SpeedVac to less than 10 µl, and desalted
using a Zip-Tip (Millipore, Bedford, Mass.). An aliquot of the digest
was mixed with an equal volume of matrix material
( Immunoblot analysis.
Polyclonal antibodies to phosphorylated
T81 (boldface) were generated (Research Genetics) in rabbits against
phosphopeptide PAPAAPTPAAPAP. Serum was
precleared on a nonphosphorylated peptide followed by affinity
purification on the phosphorylated peptide. Immunoblot analysis with
phospho-T81 antibody was performed (using a dilution of 1/100) on
proteins that were prenormalized with respect to total p53 levels
followed by enhanced chemiluminescence detection (Amersham). In all
other cases Western blotting was performed using equal amounts of
proteins. Immunoprecipitations were carried out on 1-mg quantities of
cellular extracts using the relevant antibodies and protein G beads
(GIBCO-BRL).
Confocal immunofluorescence.
Cells grown on
22-mm2 coverslips were fixed in freshly prepared 2.5%
para-formaldehyde in phosphate-buffered saline (PBS) (pH 7.4) for 10 min at room temperature. The cells were then washed three
times (5 min each) in PBS followed by permeabilization in 0.1% Triton
X-100 in PBS (pH 7.4) for 5 min and an additional three 5-min washes in
PBS. The cells were incubated on 75-µl drops of anti-p53 antibodies
(FL-393; Santa Cruz Biotechnology) diluted (200 ng/ml) in PBS (pH 7.4)
containing 1% bovine serum albumin (BSA) for 1 h at room
temperature in a humidity chamber. The cells were washed three times in
PBS (5 min each) before incubation with 75-µl drops of
fluorescein-conjugated anti-rabbit immunoglobulin G (Vector
Laboratories) diluted (15 µg/ml) in PBS (pH 7.4) containing 1% BSA
for 1 h at room temperature in a humidity chamber in the dark. The
cells were washed three times in PBS (5 min each), followed by a brief
wash in 0.1% Triton X-100 in PBS, pH 7.4 (5 min), and three additional
5-min washes in PBS, and the coverslips were mounted on glass slides in
Vectashield (Vector Laboratories).
Transcriptional activation and growth inhibition assays.
The
Mdm2 and p21CIP1/WAF1 minimal
promoter-driven luciferase constructs (cotransfected with
Apoptosis and cell cycle studies.
p53 null cells were
exposed to treatments 24 h after transfection. Apoptosis was
assessed by annexin-V-fluorescein isothiocyanate staining in the
presence of propidium iodide (PI) (Pharmingen). Cell cycle analysis of
green fluorescent protein (GFP)-positive cells was carried out using
fluorescence-activated cell sorter analysis of PI-stained cells (5 × 104 cells per sample) using a Calibur flow cytometer
(Becton Dickinson) under wide-scatter gates. The data were analyzed
using CellQuest software.
Mass spectrometry analysis of p53 phosphorylation by JNK.
Human p53HA immunopurified from
JNKKCAA-expressing 293T cells was separated by SDS-PAGE and
digested in situ with trypsin. Analysis of the peptide digests by mass
spectrometry identified a peptide mass (m/z) of
3528.5 from the p53/JNKK+ isolate that was not detectable
in the p53/JNKK T81 is phosphorylated by JNK.
Immunopurified JNK
phosphorylated the wt p53 but not the mutant form of p53 in which T81
was replaced with Ala (T81A) (Fig. 1a).
To detect T81 phosphorylation in vivo, we raised antibodies to a
phosphorylated T81 peptide. Recognition by phospho-T81 antibodies was
inhibited by a phosphorylated, but not by a nonphosphorylated, peptide
corresponding to this domain (Fig. 1b). Coexpression of the
constitutively active forms of MEKK1 (
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.8.2743-2754.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Jun NH2-Terminal Kinase Phosphorylation
of p53 on Thr-81 Is Important for p53 Stabilization and Transcriptional
Activities in Response to Stress

![]()
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
![]()
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
![]()
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
P7) and the
constitutively active forms of JNKK2 and MEKK1 kinases have been
previously described (18). FLAG-tagged MKP5 expression
vector was constructed by amplifying the MKP5 open reading frame using
reverse transcription-PCR and subcloning the amplification product into
a pcDNA3.0 plasmid (Invitrogen). A monoclonal antibody directed against
MKP5 was raised against a fragment spanning aa 2 to 154 of MKP5 (A. Bar Shira et al., unpublished data).
-32P]ATP (50 cpm/fmol) in the presence of kinase buffer. The reaction mixture was
then separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and subjected to Coomassie blue staining to
confirm equal loading of p53. The gel was dried, and phosphorylation of
p53 was determined by autoradiography. In all cases the buffers used
contained a cocktail of protease and phosphatase inhibitors
(1).
-cyano-4-hydroxy-cinnamic acid) (Sigma) as a saturated solution in
50% acetonitrile-0.05% TFA, and ion masses were determined by
matrix-assisted laser desorption-time of flight mass spectroscopy
(42) (Kratos; KOMPACT MALDI III). The instrument was
calibrated over the range 905.05 to 5,734.59 atomic mass units using
peptide mass standards (PerSeptive Biosystems).
-galactosidase [
-Gal] expression vector) were analyzed 24 h after transfection as previously described (18). Growth
inhibition assays were performed in six-well plates using H1299 cells
transfected with wt or mutant p53T81A. Cells (triplicate
wells) were subjected to G418 selection for 3 weeks before clones were
stained and counted.
![]()
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
digest. Conversely, a peptide mass
(m/z) of 3446.2 was found in the digest of the
p53/JNKK
sample but was not detected in the
p53/JNKK+ digest. A theoretical tryptic digest of p53
predicts a unique m/z of 3445.97 corresponding to
peptide 66 to 101. Within the 0.1% error of the mass spectrometer, our
results are consistent with a single phosphorylation of p53 peptide 66 to 101 (3445.97 + 80 = 3525.97) caused by coexpression of p53 with
JNKKCAA. Subsequent analysis was carried out on T81, which
is the only residue within this region that is a proline-directed
Ser/Thr, a conserved JNK phosphoacceptor site. Analogs of human
proline-driven T81 are found in p53 from other species, including mice
(Thr 78), rats (Thr 89), and canines (Thr 76).
MEKK1) or JNKK
(JNKK2CAA), but not MKK6 (MKK6DD), with wt p53
led to efficient phosphorylation of wt p53 detected by phospho-T81
antibodies. Neither kinase could phosphorylate the p53 T81A mutant
(Fig. 1c). These data suggest that JNK, but not p38, phosphorylates p53
at T81. In the same vein, forced expression of JNK2APF, a
dominant-negative form of JNK decreased MEKK1- as well as JNKK-mediated
phosphorylation of p53 on T81 (Fig. 1c). Expression of a 20-aa peptide
that corresponds to the JNK association site on p53 (P7) and that
disrupts the JNK-p53 complex in vivo and in vitro (16)
decreased JNKK-dependent phosphorylation of p53 on T81 (not shown).
Similarly, JNKK did not induce phosphorylation of T81 on p53 whose JNK
docking site had been deleted or mutated (18), indicating
that the association of JNK with p53 is required for its
phosphorylation at T81 (Fig. 1d). These experiments establish that JNK
phosphorylates p53 on T81.

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FIG. 1.
JNK phosphorylates p53 on T81. (a) Wild-type or mutant
(T81A) forms of human p53 were in vitro translated, purified, and
subjected to phosphorylation using JNK immunopurified from UV-treated
293T cells. Middle, immunoblot analysis of material subjected to
autoradiography using DO1 antibodies. The doublet represents the
nonphosphorylated (lower band) and phosphorylated (upper band) forms of
p53. The control reaction using c-Jun as a substrate (immunokinase
[IK] reaction of GST-Jun) and the level of HA-JNK2 that was
immunoprecipitated (IP-HA-JNK2) are shown (bottom). (b) Proteins
prepared from NHF that were exposed to sham stress (control) or heat
shock (HS; 42°C for 1 h) were subjected to immunoblot analysis
using the antibodies to phosphorylated T81 in the presence of excess
phospho or nonphospho peptides as indicated (top) or to Western
blotting using DO1 antibodies (bottom). (c) T81 phosphorylation on wt
or T81A forms of p53 was monitored after cotransfection of the
respective construct (1 µg) with the indicated upstream kinases (3 µg) into H1299 cells. Analysis of T81 phosphorylation was carried out
using the antibodies to phospho-T81. Bottom, p53 levels detected with
DO1 antibodies. (d) T81 phosphorylation of p53 impaired in JNK
association. Either wt rat p53 or p53 with deletions (
P7) or
mutations (at aa 101, 105, and 108) within the region, which
encompasses the JNK binding site (1, 18) was cotransfected
with JNK2CAA into H1299 cells. Proteins were prepared and
subjected to Western blot analysis using phospho-T81 antibodies (top)
or DO1 antibodies (bottom).
T81 is phosphorylated in vivo following exposure to DNA-damaging
agents.
To determine changes in T81 phosphorylation in response to
stress and DNA damage, NHF were exposed to heat shock,
cis-platinum, anisomycin, H2O2, or
UV treatment. Immunoblot analysis using antibodies to phospho-T81
revealed that T81 is phosphorylated in vivo within 1 h after
exposure to heat shock, hydrogen peroxide, sorbitol, or UV (Fig.
2a; please note that the level of p53 has
been normalized to ensure that changes in phosphorylation are not due
to altered expression level). The degree of JNK activation in response
to these treatments was monitored via immunokinase reactions using GST-Jun as a substrate (Fig. 2b). Treatment with
cis-platinum (Fig. 2a) or X rays (not shown) neither
activated JNK nor phosphorylated p53 at T81 in the NHF cells. Time
course analysis demonstrated that the degree of T81 phosphorylation
depends on the UV dose. Low-dose UV irradiation (10 J/m2),
which causes growth arrest, resulted in T81 phosphorylation within as
little as 30 min (not shown), with increased phosphorylation after 1 to
2 h. The level of T81 phosphorylation remained above background
even 24 h after UV treatment (Fig. 2a). In response to high-dose
irradiation (50 J/m2), which causes apoptosis,
phosphorylation on T81 was seen within 1 h, peaked within 4 h, and
declined to background levels within 24 h (Fig. 2a). The kinetics
of T81 phosphorylation was consistent with increased levels of p53 in
response to these stress-inducing agents (1, 17).
|
Substitution of T81 for Ala attenuates UV- and JNK-mediated p53 stabilization. A prerequisite for p53 ability to elicit its activities is its enhanced stability, which is phosphorylation dependent (18). The association of nonactive JNK with p53, which takes place under nonstressed conditions, targets p53 ubiquitination and degradation in an Mdm2-independent manner (17). Conversely, signaling by upstream JNKK kinase MEKK1 prolongs p53's half-life (16). To monitor the half-life of p53 mutated on T81A, p53 null cells were transfected with the wt or T81A mutant forms of p53 and pulse-chase analysis was carried out under normal growth conditions as well as after UV irradiation or JNKK expression.
Both wt and T81A forms of p53 exhibited short half-lives in the absence of stress (Fig. 3, left). This finding indicates that both forms are similarly targeted for ubiquitination and degradation. In response to UV treatment the half-life of wt p53 was prolonged, compared with the half-life of the T81A mutant (Fig. 3, middle). These data suggest that mutation in T81 is sufficient to impair the stabilization of p53 in response to UV irradiation. To directly address whether JNK's ability to alter p53 stability is impaired in the T81A mutant, p53 null (10.1) cells were cotransfected with the constitutively active form of JNKK2 (JNK2CAA) and either the wt or the T81A form of p53. Half-life measurements performed on the cells 24 h later revealed that JNKK increased the stability of the wt but not the T81A form of p53 (Fig. 3, right). Differences in the half-life of the T81A form of p53 in response to JNKK were more noticeable than those after UV treatment. This is not surprising in light of the multiple kinases induced by UV treatment, which contribute to p53 phosphorylation and stabilization, including p38 and Chks (7, 10, 21, 40, 51, 52). These observations suggest that T81 phosphorylation is required for JNK-mediated changes in p53 stability. Further support for the role of JNK in stabilizing p53 via T81 was obtained from analysis of p53 stability under cycloheximide-chase conditions (27). Exposure of cells to cycloheximide conditions (60 µg/ml) that activate JNK but block protein synthesis revealed a prolonged half-life for the wt, but not the T81A, form of the protein (data not shown). These data establish the role of T81 in p53 stabilization following UV irradiation or JNK activation.
|
Role of T81 phosphorylation in p53 transcriptional activities,
p53-mediated growth inhibition, and growth arrest.
To determine
whether T81 phosphorylation is important for activation of p53's
biological functions, we first assessed the transactivation capacities
of the T81A mutant. Basal transcriptional activities of
p21WAF1/CIP1 promoter sequences linked to a
luciferase reporter were fivefold higher for wt p53 than for the T81A
form, which exhibited activities similar to those seen with the empty
expression vector. A substantial increase (16-fold over basal levels)
in the level of p21WAF1/CIP1-mediated luciferase
activity upon expression of the constitutively active form of MEKK1
(
MEKK1) was recorded. In response to the constitutively active JNKK2
(JNKK2CAA) or after UV irradiation, the expression of the
wt but not the T81A form led to a sixfold increase in transcriptional
activities mediated by p21WAF1/CIP1 (Fig.
4a) or Mdm2 promoter sequences
(data not shown). Transcriptional activities of T81A were equal to
those of transcriptionally "dead" hot spot p53 mutants (at aa 248 and 273), before as well as after UV irradiation (Fig. 4b).
Corresponding to the level of luciferase activity driven by the
p21WAF1/CIP1 promoter are expression levels of
p21WAF1/CIP1, which were monitored via Western
blotting (Fig. 4b, bottom). These data suggest that phosphorylation on
T81 contributes to transcriptional activities of p53 in response to
stress.
|
JNK phosphatase MKP5 efficiently attenuates JNK-mediated T81
phosphorylation, p53 transcriptional activities, and p53-mediated
apoptosis.
As an independent approach to investigating the role of
JNK in T81 phosphorylation and in p53 activities, we used specific JNK/p38 phosphatase MKP5 (55). Forced expression of MKP5
in JNKK-expressing cells reduced the level of T81 phosphorylation in a
dose-dependent manner (Fig. 5a). MKP5
expression decreased, albeit less efficiently, T81 phosphorylation in
response to UV irradiation (Fig. 5a). Under these conditions, MKP5 did
not affect the level of Ser 15 phosphorylation. Differences in MKP5
effects on T81 phosphorylation in JNKK versus UV-treated cells may be attributed to the degree of MKP5 activities. Analysis of JNK
phosphorylation revealed that MKP5 was less effective in removing
phospho groups from JNK in UV-treated cells than in JNKK-expressing
ones. Accordingly, UV may attenuate MKP5 activities, which would also
explain why MKP5 was less effective in attenuating T81 phosphorylation
in UV-treated cells. Further support for the role of JNK in T81
phosphorylation and activities comes from comparison between wt and
T81A mutant forms of p53. Coexpression of JNKK and p53 (wt or T81A) in
p53 null mouse fibroblasts in the presence or absence of MKP5 revealed a clear increase in T81 phosphorylation on the wt but not the T81A
mutant form of p53, which was attenuated upon MKP5 expression. Decreased T81 phosphorylation coincided with a decrease in p21 expression (Fig. 5b). These results coincide with luciferase
assays performed under the same conditions. Expression of JNKK with p53 in p53 null cells caused a threefold increase in p53 transcriptional activities, monitored using Bax and p21 promoter sequences linked to
the luciferase reporter gene. However, addition of MKP5 to the
transfection mixture decreased p53 transcriptional activities. The
degree of decreased p53 transcription depended on the amount of MKP5
transfected (Fig. 5c). MKP5 did not have any effect on Mdm2-luc or
Bax-luc activities (data not shown). These observations further support
the role of JNK in T81 phosphorylation and transcriptional activities.
|
Cellular localization of wt and T81 p53 following JNKK and MKP5
expression.
Given the relationship between cellular localization
of p53 and its stability and activities, we monitored possible changes in p53 distribution after JNKK and MKP5 expression. As shown in Fig.
6, the expression of either JNK kinase or
its phosphatase did not alter the nuclear localization of p53. These
data suggest that JNK phosphorylation does not affect the cellular
compartmentalization of p53. A similar analysis performed on the T81A
form of p53 revealed that the lack of the JNK phosphorylation site led
to equal distribution of this p53 mutant in the cytoplasm and the
nucleus, which was not affected by MKP5 or by JNKK expression (Fig. 6,
bottom). Cellular distribution of either wt or T81A forms of p53 was
not affected by UV irradiation, nor was it changed upon leptomycin B
treatment (not shown). Since the phosphoacceptor site studied here lies within the proline-rich domain, which has been implicated in p53 conformation, we also monitored possible changes of p53 conformation due to its mutation on T81. Unlike p53 mutated on aa 248 (13), T81A p53 retained the conformation of wt p53 forms,
as determined on the basis of the peptide digest pattern (data not
shown).
|
T81 phosphorylation is attenuated in cells treated with antisense
JNK.
To further establish the role of JNK in the phosphorylation
of T81, we used antisense oligonucleotides to both JNK1 and JNK2 (JNK1AS and JNK2AS). Previous studies established that treatment of
cells with JNK-AS efficiently blocks JNK expression at the RNA and
protein levels (46), thereby creating a transient nearly null environment for JNK. Using MCF7 cells (which contain the wt form
of p53), we monitored changes in endogenous p53 phosphorylation on T81
following UV irradiation of cells pretreated with JNK1AS and JNK2AS.
Transfection of JNK1AS and JNK2AS led to a significant decrease in the
level of JNK1 and JNK2 RNA (not shown) and in protein levels (Fig.
7). Within 2 h after UV irradiation,
T81 was similarly phosphorylated in mock-, pcDNA3-, and control
antisense-oligonucleotide-transfected cells. However, a substantially
lower degree of phosphorylation was evident in the JNK1AS- and
JNK2AS-transfected cells (Fig. 7). These results establish that in vivo
phosphorylation of T81 in response to UV treatment is mediated by JNK.
|
T81 and the JNK docking site on p53 are often mutated in human cancer. A further indication of the importance of JNK phosphorylation on T81 for p53 functions comes from the finding that human tumors contain p53 with mutations within the JNK binding and phosphorylation sites. A search of the p53 mutant database for mutations within T81 identified two cases of breast and skin tumors with p53 mutated at this site. More than 180 additional tumors were found to harbor p53 with mutations on residues within, or adjacent to, the JNK docking/phosphorylation sites (aa 81 to 117), indicating that mutations occur frequently in this area (5). This finding provides additional evidence of the importance of JNK association and phosphorylation of p53.
| |
DISCUSSION |
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Increasing evidence points to the physiological importance of posttranslational modifications for p53 stability and activities. Those modifications include phosphorylation and sumoylation, which have been associated with the transcriptionally active form of p53 (21, 23, 37). Whereas p53 modification by conjugation to SUMO-1 on a single lysine residue (K386) is thought to enhance the transactivation activity of p53, multiple phosphorylation sites appear to be required for p53 stability and transcriptional activities.
The present study examines the role of in vivo phosphorylation of the newly recognized T81 site in response to various stress stimuli and shows that JNK is the phosphorylating kinase. Support for T81 as the JNK phosphoacceptor site has been provided by seven independent approaches, including (i) in vivo phosphorylation of p53 by JNKK as revealed by mass-spectrometric analysis of immunopurified p53, (ii) in vitro phosphorylation of bacterially expressed p53 by a purified form of active JNK, (iii) lack of phosphorylation by p38, (iv) inhibition of T81 phosphorylation by the dominant-negative form of JNK, (v) lack of T81 phosphorylation on p53 that lacks the JNK binding site, (vi) inhibition of JNK phosphorylation in vivo in cells that exhibit the nearly null JNK environment generated by JNK antisense oligonucleotides, and (vii) inhibition of T81 phosphorylation in vivo by JNK phosphatase MKP5.
Phosphorylation at this site is of crucial importance in relation to p53's ability to elicit transcriptional activation under conditions where JNK is activated (i.e., UV) and was abolished in p53 mutated at T81. Abrogated transactivation of p53 impaired its ability to elicit growth arrest, growth inhibition, and programmed cell death, all of which are key regulatory events in p53-mediated responses of cells to DNA damage. A lower degree of nuclear accumulation, as revealed by our confocally based immunohistochemistry analysis, may account for some, but not all, of the observed decrease in T81A p53's ability to elicit transcriptional activities of p21, Mdm2, or Bax. Whereas 50% of the T81A form of p53 is localized within the nucleus, 90% of p53 transcriptional activities were lost. Furthermore, T81A p53's loss of function also cannot be attributed to altered conformation, since peptide digestion revealed a pattern similar to that of the wt but not that of the conformationally altered 248 mutant. Lack of a significant difference in the half-life of p53 under nonstressed conditions also supports the notion that the T81A protein is under regulation similar to that of the wt counterpart.
Independent confirmation of the role of JNK phosphorylation of T81 on p53 was provided by the use of JNK1 and -2 antisense oligonucleotides and JNK phosphatase MKP5. Expression of antisense JNK1 and -2 efficiently attenuated UV-mediated T81 phosphorylation in cells. Along those lines, forced expression of MKP5 effectively reduced T81 phosphorylation following JNKK expression. A less-pronounced effect was seen in UV-treated cells, probably as result of the activation of additional p53 kinases that also contribute to p53 stability as well as to the attenuation of MKP5's activities. MKP5 was also efficient in attenuating both JNKK-mediated p53 transcriptional activity and JNKK-dependent p53-elicited apoptosis. Taken together, our data provide direct support for the finding that JNK phosphorylation of p53 takes place at T81 and that this phosphorylation is important for p53 stability and activities.
It is important to note that p53 phosphorylation by JNK is conformation sensitive; attempts to phosphorylate in vitro the GST-p53 fusion protein with JNK failed unless the GST portion of the protein was first removed (2). In this connection it is notable that an earlier study suggested that JNK phosphorylates p53, but at a residue different from that reported in the present study (38). This discrepancy may also relate to the use of murine p53; the site in question (Ser 34) has no human counterpart.
It is necessary to understand the relationship between phosphorylation on T81 and that on other sites that are phosphorylated in response to DNA damage, including Ser 15, 20, 33, 46, and 389. Growing evidence suggests that the type and dose of stress or damage incurred are important factors in determining the type of kinases induced and the pattern of p53 phosphorylation (8). It is likely that phosphorylation on certain residues may precede that on others. Accordingly, phosphorylation at one site may be required to acquire a certain conformation, enabling subsequent phosphorylation events to take place on other domains of p53. Such a model is supported by the finding that Ser 15 phosphorylation is affected by mutation of Ser 33 and Ser 46, which are p38 phosphorylation sites (7), which may explain the different kinetics that are often observed in an examination of p53 phosphorylation on different residues. Along these lines, although phosphorylation of p53 by JNK at T81 is important for p53 transactivation, it is likely to be augmented by other stress- and DNA damage-responsive kinases (e.g., ATM, p38, Chk2) that phosphorylate p53 on other residues. The requirement for coordinated phosphorylation of p53 by different kinases implies that JNK phosphorylation on T81 may not be sufficient for p53 stabilization and transcriptional activities, thereby explaining why certain JNK activators (e.g., interleukin 1) do not stabilize or activate p53 (41). Lack of JNK phosphorylation under such conditions may be overruled by other stress-inducible kinases that can phosphorylate p53 on other residues.
An important indication of the importance of T81 in p53 functions comes from the observation that close to 200 human tumors contain p53 with mutations within the domain that encompasses JNK binding and JNK phosphorylation sites. Those mutants are expected to exhibit impaired JNK association and phosphorylation and, as a result, to lack transcriptional activities.
In establishing the role of JNK and T81 in the stability and activity of p53, our study identifies an additional mechanism underlying the p53-dependent cellular response to stress and damage.
| |
ACKNOWLEDGMENTS |
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We thank Victor Adler, Karen Quadrini, and Ekaterina Matusevich for technical assistance, Roger Davis, Michael Karin, and Audrey Minden for stress kinase constructs, Xei Wu and Toru Ouchi for p53 constructs, and Hidetoshi Tahara for NHF. We also thank James Manfredi for advice on conformation analysis.
This study was supported by grant CA78419 from the National Cancer Institute (to Z.R.) and by an NIH shared instrumentation grant (1 S10 RR0 9145-01) and NSF Major Research Instrumentation grant (DBI-9 724504).
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FOOTNOTES |
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* Corresponding author. Mailing address: The Ruttenberg Cancer Center, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1130, New York, NY 10029-6574. Fax: (212) 849-2446. E-mail: zeev.ronai{at}mssm.edu.
Present address: Department of Animal Biology, University of
Pennsylvania, School of Veterinary Medicine, Philadelphia, PA 19104-6046.
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