From the Department of Molecular and Cellular
Pathology, The Cancer Research Campaign Laboratories, Dundee Cancer
Research Center, University of Dundee, Dundee DD1 9SY, United Kingdom
and the § Department of Cellular and Molecular Oncology,
Masaryk Memorial Cancer Institute, Brno 656 53, Czech Republic
Received for publication, April 24, 2000, and in revised form, October 6, 2000
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ABSTRACT |
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p53 protein activity as a transcription factor
can be activated in vivo by antibodies that target its
C-terminal negative regulatory domain suggesting that cellular enzymes
that target this domain may play a role in stimulating
p53-dependent gene expression. A phospho-specific
monoclonal antibody to the C-terminal Ser315
phospho-epitope was used to determine whether phosphorylation of
endogenous p53 at Ser315 can be detected in
vivo, whether steady-state Ser315 phosphorylation
increases or decreases in an irradiated cell, and whether this
phosphorylation event activates or inhibits p53 in
vivo. A native phospho-specific IgG binding assay was
developed for quantitating the extent of p53 phosphorylation at
Ser315 where one, two, three, or four phosphates/tetramer
could be defined after in vitro phosphorylation by
cyclin-dependent protein kinases. Using this assay,
near-stoichiometric Ser315 phosphorylation of endogenous
p53 protein was detected in vivo after UV irradiation of
MCF7 and A375 cells, coinciding with elevated p53-dependent
transcription. Transfection of the p53 gene with an alanine mutation at
the Ser315 site into Saos-2 cells gave rise to a form of
p53 protein with a substantially reduced specific activity as a
transcription factor. The treatment of cells with the
cyclin-dependent protein kinase inhibitor Roscovitine
promoted a reduction in the specific activity of endogenous p53 or
ectopically expressed p53. These results indicate that the majority of
p53 protein has been phosphorylated at Ser315 after
irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.
The cells of higher eukaryotes are subjected to multiple forms of
cellular stress, and the biochemical processes that ensure such damage
is sensed and repaired are critical for the prevention of diseases such
as cancer. Activation of p53-induced growth arrest or apoptosis is a
key end point to the signal transduction pathways induced by many
distinct forms of cellular damaging agents, including ionizing and
nonionizing radiation (1, 2), anti-metabolites that inhibit
ribonucleotide biosynthesis (3, 4), heat shock (5), hypoxia (6), and
low extracellular pH (7). The biochemical activity of p53 most
tightly linked to its biological function involves its ability to bind
to DNA sequence-specifically (8) and to function as a transcription
factor (9). p53 is composed of at least four functional domains that
regulate its activity as a transcription factor: (i) an N-terminal
trans-activation domain that is required for interaction with
components of the transcriptional machinery, including p300 (10, 11);
(ii) the central conserved core DNA-binding domain containing most of
the inactivating mutations found in human tumors (12); (iii) a
tetramerization domain (13); and (iv) a C-terminal negative regulatory
domain whose phosphorylation, acetylation, or SUMOylation correlates with activation of the latent sequence-specific DNA binding function of
p53 (14-19). Each of these domains on p53 contain multiple sites for
modification by both covalent and noncovalent interactions, and it now
seems likely that it is the combined action of many enzymes that
coordinately modulate p53-dependent gene expression in
response to cellular stress. Given that 2-fold changes in the gene
dosage of p53 can have dramatic affects on tumor incidence in
vivo (20, 21), it seems evident that these post-translational events modulating the specific activity of p53 will play an important role in regulating its tumor suppressor function.
The N-terminal regulatory domain of p53 contains a highly conserved
15-amino acid domain that directs the binding of p53 to the positive
effector p300 or the inhibitor MDM2, the balance of which modulates
p53-dependent tumor suppression (22). The regulation of the
p53-p300 interaction is modulated in part by phosphorylation within the
BOX-I domain, as p300/CBP binding to p53 in the N terminus and
subsequent acetylation of the C-terminal domain of p53 can be
stimulated by Ser15 phosphorylation within the p300 docking
site (23, 24). Consistent with these data, mutation of full-length p53
protein at multiple sites including Ser15 can reduce its
specific activity as a transcription factor in vivo (25,
26). Enhanced phosphorylation of endogenous p53 protein at
Ser15 following DNA damage, quiescence, or senescence (27)
can occur through the action of an ATM/ATR/DNA-PK
kinase-dependent pathway (28-30). These studies identified
one key signal transduction cascade that could stimulate
p53-dependent transcription via modification of the
N-terminal domain of p53.
Two other sites of post-translational modification are now known to be
clustered within this BOX-I regulatory domain, with a similar paradigm
being supported: phosphorylation of p53 within the N-terminal BOXI
domain at Thr18 or Ser20 can destabilize the
p53-MDM2 complex or stabilize p53-p300 protein interactions
(31-33).1 The
phosphorylation sites at Thr18 or Ser20 exhibit
distinct types of regulation, depending upon the context or damaging
agent. Cycling human cells with a wild-type p53 pathway constitutively
modify p53 at the Ser20 site (35, 36), and the most likely
role for Ser20 phosphorylation under these conditions is to
produce a transcriptionally competent pool of p53 protein that binds to
p300.1 These data are consistent with the requirement for
an active pool of p53 in a cycling cell to produce p21WAF1
protein (37) and provide a mechanism for producing MDM2 protein in a
cycling cell to maintain the negative feedback degradation loop (38).
Oxidant stresses can result in hypo-phosphorylation at
Ser20 (35, 36), which will presumably destabilize the
p300-p53 complex and place an excessive oxidant burden on the p53
pathway. The ionizing radiation-induced form of p53 protein is
phosphorylated at Ser20 by a Chk2-dependent
pathway (39-41), ensuring that the p300-p53 complex is stabilized and
active in an irradiated cell for the induction of
p53-dependent gene expression. The Thr18
phosphorylation can potently inhibit MDM2 binding or stabilize p300
binding,1 and the Thr18 site is phosphorylated
in human breast cancers (31), induced during senescence (27) or
transiently following ionizing radiation (33).
Flanking the tetramerization domain of p53 in the extreme C terminus is
a negative regulatory domain whose post-translational modification also
plays an important role in modulating the specific activity of p53
in vivo. One function for this regulatory C-terminal domain
is to maintain p53 protein in a latent state for specific DNA binding.
Deletion of this domain or stoichiometric phosphorylation at
Ser392 activates the latent specific DNA binding function
of p53 in vitro by an allosteric mechanism (14). Increased
steady-state phosphorylation at Ser392 of endogenous human
p53 protein in cells occurs following UV-C and X irradiation damage
(42), but whether CK2, PKR or other enzymes catalyze this reaction is
not yet known (43-45). In addition, microinjection or intracellular
synthesis of an antibody (pAb421) that binds to the C-terminal negative
regulatory domain near the phosphorylation sites can activate
p53-dependent gene expression in vivo (46-48),
suggesting that C-terminal modification can be a rate-limiting step in
stimulating p53 function in vivo. Together, these data
provide the basis for the paradigm that a signaling pathway modulating
the specific activity of p53 after cellular damage targets the
C-terminal negative regulatory domain by stimulating the latent
specific DNA binding function of the p53. Other enzymes that are now
known to target sites within the C-terminal domains of p53 and
stimulate its specific DNA binding function have also been suggested to
play a coordinated role in the damage response, including increases in
steady-state acetylation at Lys320 and/or
Lys382 by p300/CBP (16) and increases in SUMOylation at
Lys386 (17, 18).
One site of phosphorylation in the C terminus for which a role in the
stress-induced activation of p53 has not been defined is the
cyclin-dependent kinase site at Ser315.
Conventional mapping techniques have shown that the highly conserved Ser315 site is phosphorylated in cells using conventional
[32P]orthophosphate labeling methods and is a good
in vitro substrate for the G2 and S phase
cyclin-dependent kinases (49, 50). Phosphorylation of p53
at this site has been shown to significantly enhance the
sequence-specific DNA binding activity of p53 protein in
vitro (50, 51), possibly by cooperating with other modifications within the extreme C-terminal negative regulatory domain (52) or by
alternate N-terminal modifications (53). Conversely, biophysical studies have shown that phosphorylation at the Ser315 site
reverses the stabilizing and activating effects of Ser392
phosphorylation on tetramer formation (54). Elevated phosphorylation at
the cdk/cdc phosphorylation site induced by okadaic acid treatment of
cells increased the specific activity of p53 as a DNA-binding protein
from the mdm2 or p21waf1 promoters
but reduced activity from the cyclin G promoter (55). Mutation of the cyclin-dependent kinase phosphorylation
site of rat p53, Ser313, to alanine also had differential
affects on reducing or increasing the specific activity of ectopically
expressed rat p53 from distinct promoters (55). Together, these data
indicate that the Ser315 site phosphorylation can have an
inhibitory or a stimulatory role in modulating
p53-dependent transcription, depending upon the context.
Given the central role played by the cyclin-dependent
kinases in the regulation of the eukaryotic cell cycle and the evidence indicating that Ser315 modification of p53 can be
inactivating or activating, we set out to further investigate the role
played by phosphorylation of endogenous p53 protein at
Ser315 in cells with a wild-type p53 pathway. Standard
radiolabeling techniques used to monitor phosphorylation in cells
actually perturbs the base-line phosphorylation-status of p53 and
induces a p53-dependent growth arrest and will therefore
alter cyclin-dependent kinase activity (35). To circumvent
this problem, we have utilized a noninvasive approach to dissect
signaling to Ser315 and to begin to address the role of
cyclin-dependent kinases in the regulation of p53 function.
A phospho-specific monoclonal antibody to Ser315 has been
generated and characterized to demonstrate that phosphorylation of
endogenous human p53 in MCF7 and A375 cells at Ser315 is
near-stoichiometric after cellular irradiation. Mutation of the
Ser315 phosphorylation site on p53 to alanine reduces the
specific activity of p53 as a transcription factor in vivo,
and the use of the cyclin-dependent protein kinase
inhibitor Roscovitine reduces the specific activity of wild-type p53
in vivo. These data identify a regulatory site in which the
phosphorylation stoichiometry of endogenous p53 protein is established
in vivo and link enhanced Ser315 phosphorylation
by a cyclin-dependent protein kinase pathway to an increase
in the specific activity of p53 as a DNA-binding protein and
transcription factor in vivo.
Reagents, Enzymes, and Proteins--
Anti-p53 antibodies DO-1,
DO-12, BP.10, pAb421, CM5, and CM1 have been described previously (43,
56). Anti-sep70 is a monoclonal raised to the chaperone
SEP70.2 FPS392 is a
monoclonal antibody specific to p53 phosphorylated at
Ser392 and was described previously (42). For the
generation of FPS315, mice were immunized with a keyhole limpet
hemocyanin-conjugated phospho-peptide NNTSSSPO4PQPKKKPLDG
corresponding to amino acids 310-325 on human p53 (synthesized by Dr.
G. Bloomberg, University of Bristol). Monoclonal antibodies were
generated according to established procedures (57) and characterized as
described in Fig. 1 and by Ref. 58. Expression of full-length human p53
in Escherichia coli or in Sf9 insect cells
and fractionation by heparin-Sepharose chromatography or by
phosphocellulose cation exchange chromatography was performed as
described previously (59). Recombinant human cyclin A, cdk2, cyclin B,
and cdc2 were expressed in Sf9 insect cells as
individual subunits, and the holoenzyme complex was reconstituted in
the kinase reactions as indicated previously (58).
Peptide Library Phage Display to Define the FPS315
Epitope--
Enzyme-linked immunosorbent assay wells were coated with
purified FPS315 and used to select bacteriophage from libraries
containing random peptides inserted within the phage III coat protein
according to the manufacturer's protocol (New England Biolabs). Two
separate libraries were screened: PhD12 contains random linear 12mer
peptides, whereas C7C contains 7-mer cyclic peptides, the structure of
which is constrained by a disulfide bridge between two cysteine
residues at either end of the insert peptide. After two cycles of
selection and amplification, individual phage clones were screened for
FPS315 binding by enzyme-linked immunosorbent assay, and the inserts of
strongly positive clones were sequenced and depicted in Table I.
Human Cell Lines, Culture Conditions, and Cell Lysis--
A375
malignant melanoma cells have been described previously (42, 43). A375
and MCF-7 cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (Life Technologies, Inc.). For
UV-C irradiation cells were first washed with Hank's balanced salt
solution (Life Technologies, Inc.) and then irradiated in the absence
of medium using a model 2400 Stratalinker (Stratagene), before being
refed with fresh medium. Roscovitine was obtained from
Calbiochem/Novabiochem and stored at Immunochemical Methods--
Standard immunoblotting was
performed using established techniques, as described previously (43).
Immunoprecipitation of recombinant p53 protein was performed using RIPA
buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.2, 1% sodium deoxycholate, 1% Triton X-100, and 0.1% SDS) containing 1 mM benzamidine, 50 mM NaF, 5 mM
dithiothreitol. Protein G-Sepharose beads (Amersham Pharmacia Biotech)
were preblocked in 3% BSA,3
and then added to 200 µl of buffer containing 1% BSA, with 1 µg of
antibody, and 100 ng of p53 (Heparin fraction II; Ref. 59). Reactions
were incubated for 18 h at 4 °C, and then beads were washed
extensively before being boiled in Laemmli buffer and analyzed by
Western blotting. For immunoprecipitation of p53 from cell lysates DO-1
was first cross-linked to protein G-Sepharose beads using
dimethylpimylimidate (Sigma) (57), and noncross-linked antibody was
then eluted with 0.1 M glycine, pH 2.5. Immunoprecipitation was allowed to proceed for 3 h at 4 °C. Luminographic
quantitation of p53 protein levels using enzyme-linked immunosorbent
assay was performed as indicated (58).
Sequence-specific DNA Binding Assays--
Electrophoretic
mobility shift analysis was performed differently depending on whether
recombinant protein or cell line nuclear extracts were being assayed.
Recombinant proteins were assayed at 4 °C in 10 µl of a buffer
containing 15% glycerol, 25 mM HEPES, pH 7.6, 10 mM KCl, 0.02% Triton X-100, 1 mg/ml BSA, 5 mM
dithiothreitol, 1 mM MgCl2, 1 mM
benzamidine, 100 ng of sonicated herring sperm DNA, and 1 ng of
radiolabeled PG or p21 consensus oligonucleotide (50, 62). For the
assay of nuclear lysates 5 µg (4 µl) of protein in nuclear lysis
buffer B (20% glycerol, 20 mM HEPES, pH 7.6, 1.5 mM MgCl2, 0.1% IGEPAL CA-630, 5 mM dithiothreitol, 1 mM benzamidine) was added
to a 30 µl of a buffer containing 20 µl of cytoplasmic lysis buffer
A (as buffer B, but with 10 mM KCl, 1 mg/ml BSA, and 100 ng
of sonicated herring sperm DNA). Reactions were allowed to assemble for
10 min at 22 °C before the addition of 1 ng of radiolabeled p53CON
oligonucletide (GGACATGCCCGGGCATGTCC) and the indicated monoclonal
antibody. Reaction products were processed using native polyacrylamide
gel electrophoresis as indicated previously (62). The native gel
agarose band shift assay was performed as indicated (56) with the
following modifications. Briefly, latent p53 protein (460 ng) was
incubated in a buffer containing 5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 50 mM KCl, 0.01% Triton X-100, 5 mM MgCl2, and 1 mM ATP without or
with the indicated kinases (PKC or cyclin A-cdk2) or monoclonal
antibody. BP.10 was used in place of pAb421 as the C-terminal
activating antibody with the minimal epitope defined as being
Gln375-Ser376-Thr377-Ser378
(56). Following an incubation at 30 °C for 60 min, 1 µg of pPGM-1
plasmid DNA restricted with PvuII (to give a large vector fragment and a smaller 474-base pair DNA fragment containing the p53
consensus site PG, which was originally cloned into the
HindIII site of pBluescript; Ref. 62) was added to the
reaction and the products were electrophoresed on a 0.8% agarose gel
in 1× TBE (89 mM Tris borate, 2 mM EDTA) at 10 V/cm for 200 min at 4 °C. After electrophoresis, the gels were
stained with ethidium bromide (1 µg/ml) for 30 min, rinsed in water,
and photographed.
Kinase Reactions--
Phosphorylation of E. coli-expressed human p53 was performed at 30 °C in kinase
buffer (10% glycerol, 25 mM HEPES, pH 7.6, 0.05 M KCl, 0.5 mg/ml BSA, 1 mM NaF, 1 mM benzamidine, 5 mM MgCl2, 1 mM dithiothreitol, 250 µM to 1 mM
ATP). For radioactive kinase assays [ The Development of an Immunochemical Assay to Quantitate in Vitro
Ser315 Phosphorylation of p53--
Phospho-specific
antibody reagents have recently proved to be valuable noninvasive
reagents for the study of signal transduction pathways that target p53
in vivo, especially because conventional 32P
labeling methods commonly used to identify phosphorylation sites induce
a p53-dependent growth arrest and alter phosphorylation of
p53 protein in normal human diploid fibroblasts (35). Given that
cyclin-dependent kinase activity will change after such
cell damage, the development of a noninvasive probe to this known
cyclin-dependent protein kinase site on p53 is important in
beginning to define an accurate role for this pathway in modulating p53 function.
The monoclonal antibody described in this study specific for
phospho-Ser315 (named FPS315) was raised against the
keyhole limpet hemocyanin-conjugated phospho-peptide
NNTSSSPO4PQPKKKPLDG corresponding to amino
acids 310-325 on human p53, and its use in a Cdk2 and Cdc2 kinase
assay has been reported (58). Purification of the monoclonal antibody
from hybridoma tissue culture supernatants using protein A-Sepharose
was required to obtain pure IgG devoid of contaminating phosphatases
found in serum supernatant, and this highly purified IgG was used in the in vitro assays described below. Phage peptide display
libraries were first used to define the essential residues within the
FPS315 epitope (Table I). This assay was
developed because unpublished data from our lab have shown that the
specificity of some phospho-antibodies generated to N-terminal
phosphorylation sites on p53 can be toward phospho-amino acids rather
than phospho-epitopes. Given the widespread growing use of
phospho-specific monoclonal and polyclonal antibodies, a rigorous
characterization is therefore required to ensure the integrity of the
phospho-specific IgG.
Positive peptide phage clones selected by two rounds of biopanning that
bound to the monoclonal antibody FPS315 all contained an invariant PQP
motif corresponding to
Pro316-Gln317-Pro318 on human p53;
however, differences were observed in amino acids flanking the PQP
motif depending upon the combinatorial library utilized. The linear
12-mer peptide library gave rise to a predominant set of clones with a
stabilizing glutamate (negatively charged phosphate mimetic) at the
position of Ser315 and two other clones with a glycine or
serine at the position of Ser315 (Table I). The cyclic
7-mer peptide library gave rise to clones without the expected
stabilizing aspartate at the position of Ser315 but yielded
a stabilizing Lysine at position Lys320 that was not
observed using the linear combinatorial library. Although the
differences in the affinity between each of these peptide epitope
clones are not known, these experiments demonstrate that an important
recognition feature of the monoclonal antibody FPS315 involves its
specificity for amino acids flanking the Ser315
phosphorylation site of p53 and that this antibody is therefore not
just detecting a phospho-serine moiety.
Denaturing immunoblots of human recombinant p53 protein demonstrate
that FPS315 only recognizes p53 phosphorylated at Ser315
(Fig. 1A, top
panel, lane 2) but does not recognize unphosphorylated p53 (Fig. 1A, top panel, lane 1) or
Ser392 phosphorylated p53 (Fig. 1A, top
panel, lane 3). A duplicate immunoblot was probed with
antibody DO12 to demonstrate that the p53 protein levels are equal in
all three lanes (Fig. 1A, bottom panel). FPS315
also displayed a striking specificity for the native phospho-Ser315 p53 protein because immunoprecipitation by
this antibody is dependent on the prior phosphorylation of p53 at
Ser315 (Fig. 1B, top panel,
lane 2), whereas FPS315 does not immunoprecipitate unphosphorylated p53 (Fig. 1B, top panel,
lane 1) or Ser392 phosphorylated p53 (Fig.
1B, top panel, lane 3). As controls for this immunoprecipitation, DO-12 antibody could bind equally well to
all three isoforms of p53 (Fig. 1B, middle
panel), whereas a control monoclonal antibody (named sep70) shows
that Ser315 phosphorylated p53 is not binding
nonspecifically to the solid phase (Fig. 1B, bottom
panel).
The most informative immunochemical assay we developed for studying
Ser315 phosphorylation of p53 was the electrophoretic
mobility shift DNA binding assay. The central feature of this assay
first takes advantage of the fact that a bivalent IgG molecule can bind
in cyclic manner to a multivalent antigen (63, 64), producing a stable
complex containing one or two IgG molecules per p53 tetramer (59).
Previous biochemical studies have also indicated that the number of
antibodies bound to one tetrameric p53-DNA complex can be quantitated
by counting the integral number of stable, intermediate IgG-p53-DNA
complexes separated by native gel electrophoresis (59). In theory, the
extent of Ser315 phosphorylation on p53 tetramers could
similarly be quantitated by determining whether zero (0%
phosphorylation), one (50% phosphorylation), or two (100%
phosphorylation) bivalent FPS315 IgG molecules are bound to and
supershift the tetrameric p53-DNA complex.
The immunoblot and immunoprecipitation (Fig. 1) showed that
unphosphorylated p53 protein expressed in E. coli is not
bound by FPS315. Consistent with this, unphosphorylated p53 protein is
not supershifted by FPS315 in a DNA binding assay (Fig.
2A, lane 1).
However, a time course of phosphorylation performed under conditions
where phosphorylation is substoichiometric began to give rise to a
supershift of the p53-DNA complex and proceeds steadily from a slower
migrating species obtained when one FPS315 IgG molecule is bound
unstably to one singly phosphorylated tetramer (Fig. 2A,
lane 1 versus lanes 3 and 4) to the more slowly
migrating complex obtained with one FPS315 IgG molecule bound stably to one doubly phosphorylated tetramer (i.e. 50%
phosphorylation of the tetramer; Fig. 2A, lane 1 versus lanes 7-9). Further phosphorylation results in the
production of three phosphates/tetramer and a corresponding increase in
the intensity of a slower migrating complex (Fig. 2A,
lane 9 versus lanes 1-8). Changing the kinase conditions
in vitro can also give rise to altering extents of
phosphorylation. Stoichiometric phosphorylation of p53 tetramers
(approaching 100%) can be observed as defined by nearly complete
supershifting of the p53-DNA complexes (Fig. 2B, lane
6 versus lanes 1-5). In addition, where ATP is limiting, ~50%
of the tetramers can be phosphorylated twice as defined by the presence
of equal amounts of unshifted p53-DNA complexes and a partially shifted
p53-DNA complexes containing one FPS315-IgG per p53-DNA complex (Fig.
2B, lane 4 versus lane 3). As a control, the
complete reaction containing high levels of ATP and kinase but in the
presence of the cdk/cdc inhibitor Roscovitine (250 µM) do
not yield an FPS315-shifted p53-DNA complex (Fig. 2B,
lane 2 versus lane 1). These data indicate that the extent
of Ser315 phosphorylation can be determined by quantitating
the number of FPS315 IgG molecules bound to native p53 tetramers and is
used as an assay to address the extent of Ser315 site
phosphorylation on endogenous p53 protein in vivo.
The p53 Protein Induced by UV Irradiation Is Stoichiometrically
Phosphorylated at Ser315--
Although FPS315 is highly
specific for its phospho-epitope, the affinity is relatively low, and
the IgG cannot be used to detect changes in endogenous p53 protein
phosphorylation by direct immunoblotting of lysates (data not shown).
As a result, p53 protein was first immunoprecipitated from lysates
isolated from cycling and irradiated cells to concentrate the p53
protein for immunoblotting. Using an immunoprecipitation assay,
increases in Ser315 phosphorylation can be detected 5 h after DNA damage (Fig. 3A, bottom panel, lane 3 versus lanes 1 and
2) under conditions where p53 protein is induced (Fig.
3A, top panel, lanes 2 and 3 versus lane 1). However, these immunoblotting data do not address
the stoichiometry in vivo, an important milestone in
determining the significance of an enzyme pathway interacting with p53
in cells. For example, if Ser315 phosphorylation of p53 was
very low (i.e. less than 1%), then the significance of this
pathway would assume less importance. However, if phosphorylation was
very high (i.e. greater than 50%) and the majority of the
p53 tetramers activated by radiation interacted at some stage with this
Ser315 site kinase, then it would define a pathway likely
to be important in affecting p53 function.
Using the native gel electrophoretic IgG binding assay, we elected to
use two distinct cell types expressing p53 protein for examining the
Ser315 phosphorylation stoichiometry of p53 synthesized
in vivo. Recombinant human p53 protein expressed in
Sf9 insect cells results in multiple isoelectric forms
representing differential phosphorylation states of p53 (31) that can
be partially separated by heparin-Sepharose chromatography into latent
and activated isoforms (65), and insect cell-expressed p53 has been
reported to phosphorylate p53 at the CDC2/CDK2 phosphorylation site
(51). As such, we first used the native gel electrophoretic IgG binding
assay to determine whether Ser315 phosphorylation of human
p53 occurring in vivo could be quantitated in this cell
system. The activated isoform of p53 protein purified on
heparin-Sepharose from Sf9 cells (Fig. 3B, lane
1) is phosphorylated at the pAb421 epitope (65) and is not shifted
by this phosphorylation-sensitive monoclonal antibody (Fig.
3B, lane 2). FPS315 supershifted the majority of
activated isoform of p53 protein expressed in Sf9 cells to
produce the species containing two FPS315-IgG per p53-DNA complex (Fig.
3B, lanes 3 and 4), indicating that
the majority of the p53 has four phosphates per tetramer. A small
proportion of the activated p53 contained two phosphates per tetramer
as defined by the faster migrating species containing one FPS315-IgG per p53-DNA complex (Fig. 3B, lanes 3 and
4). Together, these data demonstrate that in
vitro phosphorylation of bacterially expressed p53 protein (Fig.
2) or in vivo phosphorylation of p53 at Ser315
in insect cells (Fig. 3B) can be quantified using the native gel electrophoretic IgG binding assay and provided the foundation to
determine whether the stoichiometry of Ser315
phosphorylation could be quantified on endogenous p53 protein in human cells.
Exposure of the human cancer cell line MCF7 to UV irradiation caused a
clear increase in the levels of endogenous wild-type p53-DNA complexes
(Fig. 3C, lane 3 versus lane 1), and the majority of this complex was further shifted when FPS315 was added to the binding reaction (Fig. 3C, lane 4 versus lanes 3 and 2). These data indicate that the phosphorylation at
Ser315 induced by radiation in this cell line occurs on
greater than 95% of the endogenous p53 tetramers and that the majority
of wild-type p53 protein activated by UV irradiation interacts with the
Ser315 kinase pathway in vivo. A similar level
of Ser315 phosphorylation was observed in irradiated A375
cells (see below; Fig. 6, lane 6 versus lane 5). A standard
immunoblotting method was also employed to determine whether the
transiently transfected p53 gene produces a protein that is
phosphorylated at Ser315 following irradiation, because
this technique produces more p53 protein than is normally present
endogenously, and this allows the detection of p53 phosphorylation
using a direct immunoblotting assay. The transfection of a gene
encoding wild-type p53 into the p53-null cell line Saos-2 resulted in a
protein that exhibits a basal level of Ser315
phosphorylation (Fig. 3D, lane 1), and after UV
irradiation the transfected p53 protein displays a transient increase
in Ser315 phosphorylation 4, 8, and 12 h post-UV (Fig.
3D, lanes 3-5 versus lane 1), providing
additional evidence that phosphorylation of p53 occurs at
Ser315 following irradiation in vivo.
Phosphorylation of p53 at Ser315 Activates the Latent
Sequence-specific DNA Binding Activity of p53 in Vitro--
Before
proceeding to investigate whether Ser315 phosphorylation of
endogenous p53 protein is regulated in vivo, we first wished to further define the effects of phosphorylation of p53 at
Ser315 on the in vitro sequence-specific DNA
binding function of p53, because it is not clear whether modification
of this site alone is activating or destabilizing to the DNA binding
function of p53 (50, 54, 55). When unphosphorylated p53 protein
purified from a bacterial expression system is added to DNA binding
assays, the protein is inactive for DNA binding (Fig.
4B, lane 2) unless the activating antibody BP.10 is added (Fig. 4B, lane
3). The latent activity of unphosphorylated p53 could be activated
by enzymes that target the C-terminal regulatory domain including PKC
(Fig. 4B, lane 6 versus lanes 4 and 5)
and cyclin A-cdk2 (Fig. 4B, lane 9 versus lanes 7 and 8). The form of p53 protein activated by cyclin A-cdk2
(Fig. 4A, lane 4 versus lane 2) could be
supershifted by FPS315 (Fig. 4A, lane 5 versus lanes
4 and 3), further indicating that near-stoichiometric
phosphorylation has occurred on p53 in vitro by the action
of cyclin A-cdk2. Together, these data indicate that Ser315
phosphorylation can activate the sequence-specific DNA binding function
of p53, consistent with a previous report that this phosphorylation can
be stimulatory rather than inhibiting (50).
Mutation of the Ser315 Phosphorylation Site to Alanine
Reduces the Specific Activity of p53 as a Transcription
Factor--
Given that most of the p53 protein interacts with a
Ser315 kinase pathway after irradiation (Fig. 3), these
data suggest that this phosphorylation may be stimulatory because it
coincides with p53 activation in a damaged cell. As such, the
transcription activity of p53 was quantitated in the p53-null cell line
Saos-2 after cotransfection with a p53-responsive reporter gene and
either wild-type p53 or the phosphorylation mutant
p53-Ala315 gene. Although transient cotransfection of the
wild-type p53 gene gave rise to a relatively high level of reporter
activity from vectors containing the p53 consensus binding site from
the p21WAF1 or bax promoters (Fig.
5, A and B),
transient cotransfection of the p53-Ala315 gene gave rise
to lower activity from these promoters (Fig. 5, A and
B). As a negative control, the Ala341
substitution mutant, which produces a monomeric protein of low activity, gave rise to less activity than the wild-type p53 and the
Ala315 substitution mutant (Fig. 5, A and
B). Endogenous p21WAF1 protein was also induced
to a lower extent in Saos-2 cells after transfection of the
p53-Ala315 gene, in comparison with the wild-type p53 gene
(Fig. 5C), further indicating that an intact
Ser315 phosphorylation site is required for the highest
level of p53 activity.
Roscovitine Reduces the Specific Activity of p53 Protein in
Vivo--
The data in Figs. 4 and 5 suggest that phosphorylation of
p53 increases its DNA binding and transcription activity in
vivo by a Ser315 kinase-dependent pathway.
Because cyclin-dependent protein kinases are the only known
enzymes that can target this consensus cdk/cdc phosphorylation site
(50), it is predicted that inhibition of endogenous
cyclin-dependent kinase activity through the use of the
cdk/cdc inhibitor Roscovitine would reduce the specific activity of p53
in vivo. We therefore utilized Roscovitine to determine whether endogenous p53 protein activity and transiently transfected p53
protein activity would be reduced after inhibition of cdk/cdc function
in vivo. The majority of wild-type p53 tetramers in A375 cells have been phosphorylated at least twice after UV irradiation, as
defined by the extent of supershift of the p53-DNA complex by the
monoclonal antibody FPS315 (Fig. 6,
lane 6 versus lanes 5 and 3). The inclusion of
the cdk/cdc inhibitor Roscovitine at the time of irradiation prevented
much of the supershift by FPS315 (Fig. 6, lane 9 versus lane
8), indicating that this cdk/cdc inhibitor can prevent
Ser315 phosphorylation of the p53 protein after DNA damage.
It should be noted, however, that Roscovitine also produced a striking
increase in p53 protein levels in the irradiated cells (Fig. 6,
lane 7 versus lanes 4 and 1; and see below Fig.
7B), despite the fact that the
p53 protein induced was hypo-phosphorylated at Ser315.
Endogenous p53 activity in A375 cells from a p53-responsive reporter
was also tested in the absence or presence of Roscovitine to determine
whether the specific activity of p53 is reduced after cdk/cdc
inhibition. When A375 cells are transiently transfected with a
p53-responsive reporter, the basal level of p53 activity is reduced
from 4 to 8 h post drug-addition (Fig. 7A) and from 12 to 24 h, and the total levels of p53 activity began to return to
base-line levels (Fig. 7A), presumably because of
Roscovitine-metabolism and inactivation. Normalizing p53 protein levels
in the absence or presence of Roscovitine (Fig. 7, B and
C) to the total activity (Fig. 7A) gave rise to
the changes in p53 specific activity summarized in Fig. 7D.
The latter data indicate that the specific activity of p53 is
dramatically reduced by the cdk/cdc inhibitor and provide independent
evidence complimenting data in Fig. 5 for an activating role for a
cdk/cdc family member in the p53 response. Similar results were
observed by transfecting the p53 gene in Saos-2 cells and examining the
changes in the activity of p53 in the absence or presence of
Roscovitine. p53-dependent gene expression in Saos-2 cells
cotransfected with a p53-dependent luciferase reporter is reduced 4 or 8 h after Roscovitine treatment (Fig.
8A) under conditions where
transfected p53 protein levels were unaffected by the drug (Fig.
8B). Thus, the specific activity of both endogenous and ectopically expressed p53 protein can be reduced by inhibiting the
cdk/cdc pathway with the kinase inhibitor Roscovitine.
A variety of biochemical, cellular, and genetic approaches have
been developed to indicate that post-translational modification of p53
protein might be central to the control of its transactivation function. In particular, the artificial manipulation and activation of
p53 in vivo by using an antibody that mimics kinases by
targeting the C-terminal negative regulatory domain of p53 (14, 46, 48)
or an antibody that mimics N-terminal p53-BOXI domain kinases by
disrupting MDM2 binding (66) suggests that the function and stability
of endogenous p53 protein in cells is dependent upon post-translational
modification. The major issues addressed in this report are whether
Ser315 phosphorylation predominates in vivo in
cycling or damaged cells, given the conflicting data on the effects of
Ser315 phosphorylation on p53 activity. Our data
demonstrate that phosphorylation of p53 at Ser315 primes
and stimulates its latent DNA binding function in vitro and
that endogenous p53 protein is phosphorylated near-stoichiometrically at Ser315 in response to irradiation predominantly through
the activity of cdk/cdc-dependent pathway. Given that the
A375 cell line we use to dissect signaling to p53 also triggers
Ser392 phosphorylation after cell irradiation (42, 43),
these data indicate that two distinct C-terminal kinase pathways modify
p53 in response to DNA damage, consistent with a requirement for
enhanced C-terminal modification of p53 as a component of the radiation response. However, because the antibody to the Ser392
phosphorylation site cannot be used to address
stoichiometry,4 as is the case with the
antibody to the Ser315 phosphorylation site (Figs. 2 and
3), a direct comparison of the efficiency of these two kinase pathways
is not yet possible.
Studying the role of p53 phosphorylation in vivo in
controlling p53 function is complicated given that the act of
32P labeling, which is commonly used to map phosphorylation
sites, can actually induce a p53-dependent growth arrest
and alter immunoreactivity including Ser20
dephosphorylation in normal human fibroblasts (35, 36). Thus, the
development of noninvasive monoclonal antibodies provide valuable tools
for studying steady-state signaling to p53, especially for the
cyclin-dependent kinase site pathway, which can be
perturbed by cell damage. However, such phospho-specific reagents
require careful in vitro characterization prior to use,
because it cannot be assumed that an immune response to a hapten
produces a monospecific IgG. For example, monoclonal antibodies to
N-terminal BOX-I phosphorylation sites have given an insight into the
type of immune response possible in a polyclonal IgG prep to a
phospho-epitope. Although phospho-antibodies were obtained that were
apparently specific for the phospho-Ser20 or
phospho-Thr18 epitopes, there are some that display a
preference only for phospho-amino acid or for epitope containing a
stabilizing phosphate at any of the nearby phospho-acceptor sites
Ser15, Thr18, and Ser20 (31). Thus,
in this report we suggest that the definition of specificity for a
phospho-specific IgG requires the use of phage peptide display to
define the IgG protein-protein contact site and the use of an in
vitro kinase assay to determine whether the IgG is specific for
denatured and/or native full-length protein.
A second issue to address concerning post-translational modification of
p53 is not only whether it changes in response to cellular stimuli but
the extent to which sites are modified in vivo. For example,
the original 32P mapping data demonstrated the
Ser315 is an in vivo phosphorylation site (49),
and recent mass spectrometric data have shown that the
Ser315 site is modified in an ionizing irradiated cell
(67). However, both methods cannot address reliably the stoichiometry
of modification in vivo, which is the advantage of the
phospho-specific monoclonal antibody that can detect integral
phosphates on the endogenous p53 tetramer in a noninvasive manner. The
only previous report addressing the extent of modification of p53 was
the phosphorylation at Ser20 in cycling cells, which was
defined to be at least 70% of the total p53 population (36). The other
phospho-specific monoclonal antibodies developed to p53 protein at
Ser15 (36) and Ser392 (42) cannot be used to
define the stoichiometry of p53 modification in vivo,
because these antibodies are only specific for phospho-denatured p53
protein, and the native IgG binding assay cannot be used to address
phosphorylation stoichiometry (data not shown). Thus, the unique
feature of FPS315 monoclonal antibody has been that is displays
specificity for the native phospho-tetramer, thus allowing a
quantitation of the extent of phosphorylation at Ser315.
The development of the native electrophoretic ligand binding assay was
an important milestone in beginning to address the issue of
stoichiometry of phosphorylation of endogenous p53 at one of its highly
conserved phosphorylation sites.
The enhanced phosphorylation of p53 at Ser315 after
irradiation is consistent with models suggesting that C-terminal
modifications play a role in stimulating the specific DNA binding
function of p53. Additional evidence suggests that modification of the
C-terminal domain of p53 plays a role in controlling the
transcriptional activity of p53 in cells. A Cdk2 homologue has been
implicated in stimulating the specific activity of recombinant human
p53 as a transcription factor in yeast (68). In addition, multiple basic-to-hydrophobic amino acid mutations within the PKC motif (69) can
dramatically increase the specific activity of human p53 as a
transcription factor in cells, presumably by neutralizing negative
regulatory domain interaction with its binding site in the central
domain. Finally, selective mutation of murine p53 at the CK2 site can
regulate its growth suppressor function possibly via transrepression
(70, 71) and can increase its transcriptional activity in
contact-inhibited murine fibroblasts (72). These results suggest that
the structural integrity of p53 surrounding the CDC2, PKC, and the CK2
site phosphorylation sites can modulate the specific activity of p53 in
cells. Consistent with this hypothesis, one rate-limiting step in
activating the function of p53 as a transcription factor has been
determined; the form of p53 latent for sequence-specific DNA binding
(65) can be activated using the monoclonal antibody pAb421 as a
sequence-specific transcription factor using in vitro
transcription systems (73) and by microinjection into cells containing
p53-responsive reporter genes (14, 46). These results highlight the
importance of discovering signaling pathways and enzymes implicated in
site-specific modification of the C terminus of p53.
It remains unclear which enzymes might be targeting p53 at
Ser315 in vivo. Given that p53 can be modified
in vitro at Ser315 by cyclin B- and cyclin
A-dependent kinases (50) and that cyclin A-cdc2 is an key
cell cycle regulator (74-77), determining whether cyclin B-cdc2,
cyclin A-cdk2, or cyclin A-cdc2 is the major Ser315 kinase
in vivo would begin to address the pathway implicated in
stoichiometric modification at Ser315. Our preliminary data
indicate that although cyclin A-cdk2 is the major Ser315
kinase, we cannot rule out a contributory role by cyclin B-cdc2, cyclin
A-cdc2, or other enzymes that may target the SP motif. Precedents for
distinct kinases targeting the same phosphorylation site on p53 have
been reported for ATM or ATR at Ser15 (28-30) and CK2 or
PKR at Ser392 (44). In addition, the major
Ser315 kinase activity is down-regulated by irradiation
(data not shown), thus classifying the enzyme as similar to certain
cyclin-dependent kinases that are down-regulated by cell
damage. Although it is generally held that cdk2 activity is
down-regulated after DNA damage to ensure the induction of a
G1/S arrest, positive roles for cdk2 have been identified
in the radiation response. Cyclin A-cdk2 can induce the formation of
apoptotic bodies in Xenopus egg extracts, and elevated
levels of cyclin A-cdk2 correlate with radiation-induced apoptosis in
developing Xenopus embryos before the midblastula transition
(78). In addition, in irradiated thymocytes that undergo apoptosis in a
p53-dependent manner, an induction of Cdk2 accelerates
apoptosis, whereas its inhibition blocks radiation-induced cell death
(79). However, the mechanism of enhanced steady-state
Ser315 phosphorylation is not known and may involve a
coordinated change in the Ser315 kinase as well as the
antagonizing human CDC14 phosphatase that targets the
Ser315 site (34, 80). Thus, our data highlighting the
stoichiometric phosphorylation of p53 at the
cyclin-dependent kinase site in vivo and the
correlation between elevated Ser315 phosphorylation with
relatively high levels of p53-dependent transcription,
suggests the existence of a radiation-responsive kinase/phosphatase
axis that links p53 function to DNA-damage dependent checkpoint control.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C as 50 mM
aliquots in Me2SO. High salt nuclear extract of human cell
lines were performed as described (43, 60) and used for the
radiolabeled DNA binding assay, immunoprecipitation, and kinase
activity assays. Sf9 cells expressing p53 protein
were lysed by a whole cell lysis method (43). For immunoblotting, cells
were scraped off the dishes on ice-cold phosphate-buffered saline,
pelleted by centrifugation, and snap frozen. Frozen pellets were lysed
for 15 min at 4 °C in denaturing urea buffer (6.4 M urea, 0.1 M dithiothreitol, 0.05% Triton X-100, 25 mM NaCl, and 20 mM HEPES, pH 7.6) or Nonidet
P-40 nondenaturing lysis buffer (43), where indicated, and lysates were
clarified by centrifugation at 13,000 × g for 10 min.
Protein concentration was determined by the method of Bradford
(Bio-Rad), and aliquots were stored at
70 °C until required.
Transient transfections were performed as indicated (61). Plasmids used
included pCMVwtp53 (obtained by cloning wtp53 cDNA into the
BamHI site of pcDNA3.1(+)), pCMVala315p53, and
pCMVala341p53 (obtained by in situ directed mutagenesis
using the pCMVwtp53 as a template and the following primers containing the desired mutation: ala315 (F): 5'-aac aac acc agc tcc
gct ccc cag cca aag-3'; ala315 (R): 5'-ctt tgg ctg ggg
agc gga gct ggt gtt gtt-3'; ala341 (F): 5'-gag cgc ttc gag
atg gcc cga gag ctg aat-3'; ala341 (R): 5'-att cag ctc tcg
gag cgg ctc gaa gcg ctc-3'). pCMV
-gal was a gift from
Dr. Alain Puisieux (Centre Leon Berard, Lyon, France). Each plasmid (1 µg) was used in transfection assays unless differently stated. A375
cells or Saos-2 cells grown as indicated above were incubated with
concentrations of DNA-LipofectAMINE complexes according to
manufacturer's protocols and incubated for the times indicated in the
figure legends. Following cell manipulation, luciferase and
-galactosidase activity were analyzed and normalized as indicated,
or the p53 protein levels were determined by independent lysis in a
Nonidet P-40 nondenaturing buffer as indicated previously (43).
-32P]ATP was also
included in the reactions. Reaction products were then resolved by
SDS-polyacrylamide gel electrophoresis, and gels were either exposed
directly to film in the case of radioactive assays or immunoblotted
with phospho-specific antibodies as indicated (42, 58).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The FPS315 epitope
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Fig. 1.
The specificity of FPS315 for
phospho-Ser315. A, denaturing immunoblots.
p53 protein was left unphosphorylated (lane 1);
phosphorylated at Ser315 with cyclin B-cdc2 (lane
2); and phosphorylated at Ser392 with CK2 (lane
3), as indicated under "Experimental Procedures." The p53 was
processed by immunoblotting with FPS315 to measure the extent of
Ser315 phosphorylation (top panel) or DO-12 to
normalize for p53 protein levels (bottom panel). The
asterisk marks the position of p53. B, native
immunoprecipitation. p53 protein was left unphosphorylated (lane
1); phosphorylated at Ser315 with cyclin B-cdc2
(lane 2); and phosphorylated at Ser392 with CK2
(lane 3), as indicated under "Experimental Procedures."
The p53 protein was then immunoprecipitated with the indicated
monoclonal antibody (top panel, FPS315; middle
panel, DO-12, bottom panel, SEP70), the products were
immunoblotted, and the p53 protein was subsequently detected using the
polyclonal antibody CM-1. The asterisk marks the position of
p53.
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Fig. 2.
The stoichiometry of Ser315
phosphorylation on p53 tetramers can be determined using a native gel
electrophoretic phospho-specific IgG binding assay. A,
limiting phosphorylation. A kinase reaction containing bacterially
expressed, unphosphorylated p53 protein (59), reconstituted cyclin
A-cdk2 (58), and 250 µM ATP was incubated at 30 °C,
and aliquots were removed from the reaction at the indicated time
points (lane 1, no phosphorylation; lanes 2-9,
1, 2, 3, 4, 6, 8, 10, and 12 min of phosphorylation, respectively) and
stopped by adding to an equal volume of DNA binding buffer containing
radiolabeled consensus site DNA and 250 µM of the cdk/cdc
inhibitor Roscovitine at 4 °C. The extent of phosphorylation was
then determined using the DNA binding assay with the activating
antibody pAb421 and the phospho-specific antibody FPS315 added to the
reactions. The arrows indicate the mobility of
pAb421-p53-DNA complexes bound to FPS315 containing 0, 1, 2, or 3 phosphates per p53 tetramer. B, stoichiometric
phosphorylation. A kinase reaction containing bacterially expressed,
unphosphorylated p53 protein (59) and reconstituted cyclin A-cdk2 (58)
was assembled with: 250 µM Roscovitine and 250 µM ATP (lanes 1 and 2); 5 µM ATP (lanes 3 and 4); or 250 µM ATP (lanes 5 and 6). After an
incubation at 30 °C for 30 min, the reactions were stopped by adding
an equal volume of DNA binding buffer containing radiolabeled consensus
site DNA and 250 µM of the cdk/cdc inhibitor Roscovitine
at 4 °C. The extent of phosphorylation was then determined using the
DNA binding assay with the activating antibody pAb421 (lanes
1-6), and the phospho-specific antibody FPS315 was added to the
indicated reactions (lanes 2, 4, and
6). The arrows mark the mobility of
pAb421-p53-DNA complexes bound to one FPS315 IgG molecule per p53
tetramer (2 phosphates per p53 tetramer) or to two FPS315 IgG molecules
per p53 tetramer (4 phosphates per p53 tetramer).
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Fig. 3.
Irradiation induces phosphorylation at the
Ser315 site of p53 in vivo.
A, immunoprecipitation of radiation-activated p53. A375
cells were exposed to 20 Jm 2 UV irradiation and lysed at
the indicated time points (lanes 1-3, 0, 3, or 5 h
post-radiation, respectively). Lysates were then immunoprecipitated
with antibody DO-1 (chemically cross-linked to protein G-Sepharose
beads; Ref. 57), and then the samples were processed on a denaturing
SDS-polyacrylamide gel and immunoblotted with the monoclonal antibodies
DO-1 (top panel) and FPS315 (bottom panel). The
asterisk marks the position of p53. B,
quantitating Ser315 phosphorylation of p53 expressed in
insect cells. p53 protein expressed in insect cells was purified on
heparin-Sepharose to isolate the in vivo activated form of
p53 (fraction 20; Ref. 59) and then assayed for its ability to bind a
radiolabeled consensus oligonucleotide as indicated under
"Experimental Procedures." The monoclonal antibodies pAb421 and
FPS315 were added as indicated in the panel by +. The arrows
mark the migration of either p53-DNA complexes without FPS315
(lanes 1 and 2) and p53-DNA complexes bound to
one or two FPS315 IgG molecules (lanes 3 and 4)
as indicated in the panel by the arrow pointing to two
phosphates per tetramer (2 P-tetramer) or four phosphates
per tetramer (4 P-tetramer). C, quantitating
Ser315 phosphorylation of p53 in irradiated MCF7 cells.
MCF7 cells were either unirradiated (lanes 1 and
2) or exposed to 20 J/m
2 UV (lanes
3 and 4). 5 h after irradiation, p53 protein was
assayed from lysates for its ability to bind a radiolabeled consensus
oligonucleotide as indicated under "Experimental Procedures" with
the addition of the indicated antibodies pAb421 or FPS315. The
arrows mark the migration of either: p53-DNA complexes
without FPS315 (lane 3) and p53-DNA complexes bound to one
or two FPS315 IgG molecules (lane 4) as indicated in the
panel by the arrow pointing to two phosphates per tetramer
(2 P-tetramer) or four phosphates per tetramer (4 P-tetramer). Similar results quantitating Ser315
phosphorylation of p53 in irradiated A375 cells were obtained as in
described in the legend to Fig. 6. D, direct blotting can be
used to detect increases in Ser315 phosphorylation of
transfected p53 in irradiated Saos-2 cells. The p53 gene was
transfected into the p53-null cell line Saos-2, and 24 h after
transfection, the cells were untreated (lane 1) or exposed
to 20 J/m
2 UV followed by lysis 0, 4, 8, 12, 16, or
32 h post-irradiation (lanes 2-7, respectively). After
direct immunoblotting of lysates, the levels of phosphorylated p53 were
analyzed by incubation with FPS315 and processed as indicated under
"Experimental Procedures." p53 protein phosphorylated by cyclin
A-cdk2 is included as a phosphorylated control (lane 8) as
described previously (58) and is marked by the arrow.
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Fig. 4.
In vitro p53 phosphorylation at
Ser315 activates the sequence-specific DNA binding function
of p53. p53 protein was assembled into kinase and DNA binding
reactions using the agarose gel DNA binding assay as indicated under
"Experimental Procedures." A, supershifting of
cdk2-activated p53 by FPS315. Latent p53 protein purified from a
bacterial expression system was incubated (in lanes 2-7) in
a DNA binding reaction: alone (lane 2 versus lane 1), with
FPS315 only (lane 3), with cyclin A-cdk2 only (lane
4), with cyclin A-cdk2 followed by FPS315 (lane 5),
with cyclin A-cdk2 followed by DO-1 (lane 6), and with
cyclin A-cdk2 followed by FPS315 and DO-1 (lane 7). Products
were separated on an agarose gel as indicated under "Experimental
Procedures." B, activation of p53 by a monoclonal antibody
and kinases that target the C-terminal domain of p53. Latent p53
(lanes 2, 3, 5, 6,
8, and 9) or activated p53 from
Sf9 cells (lane 1) were assembled in a DNA
binding reaction with the indicated additional components: activated
p53 only (lane 1); latent p53 only (lane 2);
latent p53 and the activating antibody BP.10 (lane 3); PKC
only (lane 4); latent p53 only (lane 5); PKC and
latent p53 (lane 6); cyclin A-cdk2 only (lane 7);
latent p53 only (lane 8); and cyclin A-cdk2 and latent p53
(lane 9). Products were separated on an agarose gel as
indicated under "Experimental Procedures." The unbound consensus
site DNA, the unbound vector DNA, activated p53-DNA complexes, and
activated p53-DNA complexes bound to various IgG molecules are
indicated by the arrows.
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Fig. 5.
Mutation of the Ser315 site of
p53 reduces p53 activity in transiently transfected Saos-2 cells.
A and B, p53-dependent luciferase
activity after transfection. The p53-null cell line Saos-2 was
transiently cotransfected with the indicated p53-expression constructs
(wild-type p53, 315Ap53, or 341Ap53), the luciferase control vector
(light bar) or the (A) bax-luciferase
promoter and (B) the
p21WAF1-luciferase promoters (dark
bars). 24 h post-transfection, the levels of luciferase
activity were analyzed as indicated under "Experimental Procedures"
and plotted as p53-dependent activity (relative light
units, RLU) against the wild-type p53 gene, the
Ala315 mutant p53, and the Ala341 mutant p53.
C, quantitating p53 and p21 protein levels in Saos-2 cells.
The levels of wild-type p53 (top panel, lanes 1 and 2) and the Ala315 mutant form of p53
(top panel, lanes 3 and 4) produced
with increasing amounts of transfected DNA (1 and 2 µg, respectively)
were quantitated by Western blotting with the monoclonal antibody DO-1.
The levels of p21WAF1 protein produced by increasing
amounts of wild-type p53 (bottom panel, lanes 1 and 2) and the Ala315 mutant form of p53
(bottom panel, lanes 3 and 4) were
determined by immunoblotting with WA-1.
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Fig. 6.
The cdk inhibitor Roscovitine reduces the
extent of Ser315 site phosphorylation of p53 in irradiated
A375 cells. A375 cells were either unirradiated (lanes
1-3) or exposed to 20 J/m 2 UV (lanes
4-9) and refed with medium alone (lanes 1-6) or
exposed to 20 J/m
2 UV followed by 20 µM
Roscovitine (lanes 7-9). 5 h after irradiation, p53
protein was assayed from lysates for its ability to bind a radiolabeled
consensus oligonucleotide as indicated under "Experimental
Procedures" with the addition of the indicated antibodies pAb421 or
FPS315. The arrows mark the migration of either: p53-DNA
complexes without FPS315 (lanes 5 and 8) and
p53-DNA complexes bound to one or two FPS315 IgG molecules (lanes
6 and 9) as indicated in the panel by the
arrow pointing to two phosphates per tetramer (2 P-tetramer) or four phosphates per tetramer (4 P-tetramer).
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Fig. 7.
The cdk inhibitor Roscovitine reduces the
specific activity of endogenous p53 in A375 cells. A,
p53-dependent luciferase activity. A375 cells were
transfected with a p53-responsive reporter derived from the
p21WAF1 promoter as indicated under
"Experimental Procedures." 24 h after transfection, the cells
were treated with a Me2SO control or Roscovitine (40 µM) and lysed for analysis of luciferase activity at the
indicated times post-drug addition (4, 8, 12, and 24 h). The
activity is plotted as p53 activity (in relative light units,
RLU) as a function of time after addition of Roscovitine.
B, p53 protein levels in A375 cells after treatment with
Roscovitine. A375 cells were transfected with a p53-responsive reporter
as in Fig. 7A, and 24 h after transfection, the cells were treated
with a Me2SO control or Roscovitine (40 µM)
for the indicated times post-drug addition (4, 8, 12, and 24 h).
Qualitative changes in p53 protein levels from detergent lysed cells
were analyzed by standard immunoblotting procedures using the antibody
DO-1 (top panel, Immunoblot) and quantitative
changes in p53 protein levels were analyzed using a two-site capture
enzyme-linked immunosorbent assay by luminographic methods
(bottom panel, Luminography). C,
chemiluminescent quantitation of p53. The raw quantitative data derived
from luminographic analysis of p53 protein levels in Fig. 8B
(bottom panel) were quantitated in an ascent fluoroscan
luminometer, and the relative levels of p53 protein (relative light
units, RLU) are plotted as a function of time after addition
of Roscovitine. D, the specific activity of p53. The total
levels of luciferase activity quantitated in A were
normalized to total p53 protein levels in C, and the data
are plotted as p53-dependent activity (in relative light
units, RLU) as a function of time after addition of
Roscovitine.
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Fig. 8.
The cdk inhibitor Roscovitine reduces the
specific activity of p53 in transiently transfected Saos-2 cells.
A, p53-dependent Luciferase activity. The
p53-null cell line Saos-2 was transiently cotransfected with the
indicated p53-expression constructs (wild-type p53 (black
bars) and the Ala315 mutant p53 (white
bars)) and the p21WAF1-luciferase promoter.
24 h after transfection, the cells were treated with a
Me2SO control or Roscovitine (40 µM) and
lysed for analysis of luciferase activity at the indicated times
post-drug addition (4 and 8 h). The activity is plotted as
wild-type p53 and the Ala315 mutant p53 activity (in
relative light units, RLU) as a function of time after
addition of Roscovitine. B, p53 protein levels in
transfected Saos-2 cells after treatment with Roscovitine. Saos-2 cells
were transiently cotransfected with the indicated p53-expression
constructs (wild-type p53) and the
p21WAF1-luciferase promoter. 24 h after
transfection, the cells were treated with a Me2SO control
or Roscovitine (40 µM) and lysed for analysis of p53
protein levels at the indicated times post-drug addition (4 and 8 h) using standard immunoblotting procedures with the antibody
DO-1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by Grants IGA MH CR 6404-3/2000 and IGA MH CR 4783-3/2000 from the Czech Republic.
Supported by grants from the United Kingdom Medical Research
Council, the Cancer Research Campaign, Tenovus Scotland, and Moravian
Biotechnologies. To whom correspondence should be addressed: Tel.: 44-1382-496-430; Fax: 44-1382-633-952; E-mail:
T.R.Hupp@dundee.ac.uk.
** Supported by Grant GACR 312/99/1550 from the Czech Republic and EMBO Fellowship AST 9443.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M003485200
1 Dornan, D., and Hupp, T. R. (2001) EMBO Rep., in press.
2 A. Beltran, and T. R. Hupp, unpublished observations.
4 J. P. Blaydes and T. R. Hupp, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: BSA, bovine serum albumin; PKC, protein kinase C.
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REFERENCES |
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2. | Lu, X., and Lane, D. P. (1993) Cell 75, 765-778[Medline] [Order article via Infotrieve] |
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