Oligomerization Is Required for p53 to be Efficiently
Ubiquitinated by MDM2*
Carl G.
Maki
From the Harvard School of Public Health, Department of Cancer Cell
Biology, Boston, Massachusetts 02115
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ABSTRACT |
Wild-type p53 is degraded in part through the
ubiquitin proteolysis pathway. Recent studies indicate that MDM2 can
bind p53 and promote its rapid degradation although the molecular basis for this degradation has not been clarified. This report demonstrates that MDM2 can promote the ubiquitination of wild-type p53 and cancer-derived p53 mutants in transiently transfected cells. Deletion mutants that disrupted the oligomerization domain of p53 displayed low
binding affinity for MDM2 and were poor substrates for ubiquitination. However, efficient MDM2 binding and ubiquitination were restored when
an oligomerization-deficient p53 mutant was fused to the dimerization
domain from another protein. These results indicate that
oligomerization is required for p53 to efficiently bind and be
ubiquitinated by MDM2. p53 ubiquitination was inhibited in cells
exposed to UV radiation, and this inhibition coincided with a decrease
in MDM2 protein levels and p53·MDM2 complex formation. In contrast,
p53 dimerization was unaffected following UV treatment. These results
suggest that UV radiation may stabilize p53 by blocking the
ubiquitination and degradation of p53 mediated by MDM2.
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INTRODUCTION |
Inactivation of p53 is considered an important step in the
development of many human cancers. It is therefore important to determine how p53 levels are normally regulated and how this regulation is altered in cancer. The activity most associated with the tumor suppressor function of p53 is its ability to bind DNA and activate gene
transcription (1-3). This activity increases in response to DNA damage
because of stabilization of the p53 protein (4, 5). The effect of
increasing p53 is to stop cell proliferation, either through a
G1-phase cell cycle arrest or apoptotic cell death (6, 7).
Current models suggest that p53 is part of a DNA damage checkpoint
pathway that monitors the genome integrity and protects cells from
accumulating genetic damage. p53 carries out this function by
temporarily halting cell proliferation to allow DNA repair, or, in the
case of irreparable damage, by inducing cell death through apoptosis.
Two proteolytic pathways have been implicated in p53 degradation: the
calpain proteolytic pathway (8, 9) and the ubiquitin proteolytic
pathway (10, 11). The relative contribution of these two pathways to
p53 turnover has not been clarified. The hallmark of the ubiquitin
pathway is the covalent attachment of ubiquitin (an 8-kDa protein) to a
substrate, which "marks" that substrate for degradation
(reviewed in Ref. 12). Ubiquitin is first sequentially transferred
through a series of ubiquitin system enzymes, designated E1, E2, and
E3. The E3 enzyme then transfers the ubiquitin molecule to one or more
lysine residues in the substrate (13). Multiple ubiquitins are attached
to one another to form ubiquitin chains, and the multi-ubiquitinated
substrate is degraded by the 26 S proteasome. Proof that p53 is a
ubiquitin-system target came with the demonstration of wild-type
p53:ubiquitin conjugates in vivo (11). Factors that regulate
and/or participate in p53 ubiquitination have not been fully characterized.
Increasing evidence suggests that MDM2 can regulate p53 stability. MDM2
forms an autoregulatory feedback loop in which p53 activates MDM2
transcription, and increased levels of MDM2 protein then bind p53 and
inhibit its transcriptional activity (14, 15). DNA-damaging agents that
stabilize p53 can promote p53 phosphorylation at serines 15 and 37 (16). Interestingly, MDM2 was unable to inhibit the activity of p53
when p53 was phosphorylated at these sites. This could result if MDM2
is unable to bind p53 phosphorylated at Ser-15 and Ser-37. Other
studies demonstrated that MDM2 can promote p53 degradation in transient
transfection assays (17, 18). Though the basis of this degradation was not clarified, Honda et al. (19) reported that MDM2 could
promote p53 ubiquitination in vitro in the presence of E1
and E2 ubiquitin system enzymes. Further, MDM2 formed a thioester bond
with ubiquitin, a characteristic of E1, E2, and E3 enzymes. Mutations
in MDM2 that abrogated thioester bond formation also abrogated p53
ubiquitination, suggesting that MDM2 functions as an E3 enzyme in p53
ubiquitination. Taken together, these studies provide a provocative
model for the regulation of p53 stability. According to this model,
MDM2 binds p53 under normal conditions and promotes its ubiquitination and subsequent degradation by the proteasome. In response to DNA damage, p53 is phosphorylated at Ser-15 and Ser-37, preventing MDM2
binding and thus stabilizing p53. The stabilized p53 can then promote
transcription of its downstream target genes, resulting in cell cycle
arrest or apoptosis.
This report demonstrates the presence of p53:ubiquitin conjugates in
cells transiently transfected with p53 expression DNAs. High levels of
p53 ubiquitination were observed when cells were cotransfected with
MDM2, providing evidence that MDM2 can promote p53 ubiquitination
in vivo. Deletion mutants that disrupted the oligomerization
domain of p53 had low MDM2 binding affinity and were poor substrates
for ubiquitination. However, efficient MDM2 binding and ubiquitination
were restored when oligomerization-deficient p53 mutants were fused to
a heterologous dimerization domain. Finally, p53 ubiquitination was
inhibited in UV-irradiated cells, and this coincided with decreased
MDM2 protein levels and p53·MDM2 complex formation. UV radiation may
stabilize p53 by inhibiting the ubiquitination of p53 mediated by MDM2.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Expression DNAs encoding wild-type p53, mutant
p53s, and Myc-tagged ubiquitin were from Peter Howley (Harvard Medical
School). HA-tagged p53 expression DNA was from Christine Jost (Dana
Farber Cancer Institute). DNAs encoding p53-(1-363) and -(1-333) (20) were from Eric Stanbridge (University of California-Irvine). DNAs encoding p53-(1-353) and p53-333CC (3) were from Jennifer Pietenpol (Vanderbilt University). Human MDM2 expression DNA was from Steve Grossman (Dana Farber Cancer Institute).
Cell Culture and Transfections--
Human osteosarcoma cell
lines Saos-2 (p53-null) and U2OS (wild-type p53) were grown in
Dulbecco's modified Eagle's media
(DMEM)1 supplemented with
10% fetal bovine serum and 100 µg/ml each of penicillin and
streptomycin. Transfections were done using the calcium-phosphate
method (5) in 60- or 100-mm dishes with cells approximately 80%
confluent. For 60-mm dishes, 4-5 µg of each p53 plasmid was
transfected with an equal quantity of MDM2 plasmid, and the final DNA
amount was adjusted to 12-15 micrograms with herring sperm DNA. For
100-mm dishes, 10 µg of p53 plasmid was transfected with 10 µg of
MDM2, and the final DNA amount was adjusted to 25 micrograms. Saos-2
cells were washed twice with DMEM minus serum 6-8 h after addition of
the DNA precipitate and then refed with DMEM plus 10% fetal bovine
serum. Cell extracts were prepared 30-36 h later. U2OS cells were
washed and refed 16 h after addition of the DNA precipitate. Where
indicated, U2OS cells were UV irradiated (5) and extracts prepared 3-7
h later.
SDS-PAGE, Western Blots, and Immunoprecipitations--
For
detection of p53:ubiquitin conjugates, cells were rinsed with
phosphate-buffered saline and scraped into 800-µl
radioimmunoprecipitation buffer (RIPA) (2 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1.0% Nonidet P-40,
1.0% deoxycholate, 0.025% SDS, 1 mM phenylmethylsulfonyl fluoride). Cells were then sonicated for 10 pulses at setting 5, 50%
output, with a Branson 450 sonifier, and spun at 15,000 × g for 15 min. One-twentieth (40 µl) of the resulting
supernatant was examined directly by immunoblotting. p53 was
immunoprecipitated from the remaining supernatant using the p53
antibody Ab-6 (Oncogene Science). For co-immunoprecipitation
experiments, cells were rinsed with phosphate-buffered saline, scraped
into 750 µl of lysis buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride), and placed on ice for 30 min. Cells were then spun at 15,000 × g for 15 min,
and the supernatant was immunoprecipitated with either the p53 antibody
1801 (Oncogene Science) or the anti-HA polyclonal antibody HA.11
(Babco). Immunoprecipitates from RIPA or lysis buffer were resolved by
SDS-polyacrylamide gel electrophoresis and transferred to an
Immobilon-P membrane. For detection of p53:ubiquitin conjugates, the
membrane was autoclaved in water for 15 min prior to blocking with milk
and probing with the p53 antibody Ab-6. MDM2 was detected in p53
immunoprecipitates using the MDM2 polyclonal antibody N-20 (Santa Cruz Biotechnology).
 |
RESULTS |
MDM2 can promote p53 degradation in transfected cells (17, 18).
The purpose of this study was to determine the basis for this
degradation and the amino acid determinants in p53 required for this
effect. Saos-2 cells (p53 null) were transfected with expression DNAs
encoding p53, MDM2, and myc-tagged ubiquitin. p53 was then
immunoprecipitated and examined by immunoblotting with the p53 antibody
Ab-6 (Fig. 1A). A ladder of
protein bands with molecular weights larger than p53 were recognized
when p53 was coexpressed with MDM2. These bands ranged in size from
~63 to 90 kDa, consistent with the addition of one to five ubiquitin moieties to p53. When Myc-tagged ubiquitin was coexpressed with p53 and
MDM2, these bands shifted to a slightly larger molecular weight,
consistent with the additional Myc-tag. To confirm these bands as
p53:ubiquitin conjugates, cells were transfected with epitope-tagged
p53 (HA-p53) and Myc-tagged ubiquitin. p53 was then immunoprecipitated
with an anti-HA antibody and examined by immunoblotting for the myc
epitope. The myc antibody recognized a ladder of bands in the
immunoprecipitates, and these same bands were recognized by the p53
antibody Ab-6 (Fig. 1B). These results prove that the ladder
of bands is ubiquitinated p53 and thus prove that MDM2 can promote p53
ubiquitination.

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Fig. 1.
MDM2 promotes p53 ubiquitination in
transfected cells. A, Saos-2 cells (p53 null) were
transfected with DNAs encoding wild-type (wt) p53, MDM2, and
Myc-tagged ubiquitin (myc-Ub) as indicated. 40 h after
transfection, cell lysates were made in RIPA buffer. p53 was
immunoprecipitated with the p53 antibody Ab-6 and examined by
immunoblotting with Ab-6. The ladder of bands is ubiquitinated p53. The
IgG heavy chain and nonubiquitinated p53 co-migrate at the position of
the asterisk (*). The band in the mock-transfected lane is only the IgG
heavy chain. B, cells were transfected with HA-p53, MDM2,
and Myc-tagged ubiquitin as indicated, and cell lysates were made in
RIPA buffer. P53 was immunoprecipitated with the HA polyclonal antibody
HA.11 and examined by immunoblotting with the myc-antibody 9E10. The
blot was stripped and reprobed with the p53 antibody Ab-6. The ladder
of bands is ubiquitinated p53.
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Cancer-derived p53 point mutants were also tested for MDM2-mediated
ubiquitination (Fig. 2). The p53 mutants
V143A and R248W were ubiquitinated to approximately equal levels when
coexpressed with MDM2, while the R273H mutant was ubiquitinated to a
much lesser extent. R273H ubiquitination could be seen on long gel exposures, but the extent of R273H ubiquitination was consistently lower than that of either V143A or R248W. This suggests that specific p53 mutations can affect the susceptibility of p53 to MDM2-mediated ubiquitination. The relative ability of each p53 mutant to bind MDM2
was assessed to determine whether the lower ubiquitination of R273H was
because of a decreased ability to bind MDM2. This involved
immunoprecipitation of mutant p53 from transfected cells, followed by
immunoblotting for MDM2 (Fig. 3).
Approximately equal levels of MDM2 were detected in V143A and R273H
immunoprecipitates, while slightly lower MDM2 levels were detected in
R248W immunoprecipitates. These results suggest that the decreased
ubiquitination of R273H is not because of a decreased ability to bind
MDM2.

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Fig. 2.
Ubiquitination of p53 mutants. Saos-2
cells were transfected with DNAs encoding the indicated p53 point
mutant alone, or cotransfected with MDM2.
p53-Ubn, nonubiquitinated p53. A, 100 micrograms of cell extract were examined by Western blotting with the
p53 antibody Ab-6. B, the blot in panel A was
stripped and reprobed with the MDM2 antibody N-20.
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Fig. 3.
MDM2 binding by mutant p53s. Saos-2
cells were transfected with the indicated p53 mutant alone, or
cotransfected with MDM2, and cell lysates were prepared. A,
p53 was immunoprecipitated with the p53 antibody 1801, and the
immunoprecipitates were examined by immunoblotting with the MDM2
antibody N20. B, 50 micrograms of protein extract were
examined without prior immunoprecipitation by immunoblotting for p53
and MDM2.
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Deletion analysis was used to identify p53 regions required for
MDM2-mediated ubiquitination (Fig. 4).
p53 contains four main functional domains (21). Amino acids 1-42
comprise the transcriptional activation domain, and it is this region
that binds MDM2. Residues102-292 encode the sequence-specific DNA
binding domain. p53 binds DNA as a tetramer, and oligomerization is
mediated by residues 324-355. The carboxyl terminus of p53 (residues
367-393) binds DNA nonspecifically and can allosterically regulate DNA
binding by the central region. p53 proteins lacking 30 (p53-(1-363)),
40 (p53-(1-353)), or 60 (p53-(1-333)) C-terminal amino acids were
tested for MDM2-mediated ubiquitination. p53-(1-363) and p53-(1-353)
maintain the oligomerization function of p53, while oligomerization is
lost in p53-(1-333) (22). All three deletion mutants maintain the
major nuclear localization signal (NLSI, residues 316-325) of p53
(23). Wild-type p53 and the deletion mutants p53-(1-363) and
p53-(1-353) were efficiently ubiquitinated by MDM2 (Fig. 4),
indicating that sequences between 353 and 393 are not critical for
ubiquitination. In contrast, ubiquitination was drastically reduced in
p53-(1-333), indicating that sequences between 333 and 353 are
required for efficient ubiquitination. There are three possible
explanations for these results. First, deletion of residues 333 to 353 may have removed critical lysine residues that are sites of p53
ubiquitination. There is one lysine (position 351) between residues 333 and 353, and a p53 protein with this site mutated to isoleucine was
efficiently ubiquitinated by MDM2 (not shown). Second, deletion of
residues 333 to 353 may have abrogated MDM2 binding. Third, p53
oligomerization may be required for ubiquitination by MDM2, and
deleting this function in p53-(1-333) may have thus prevented
ubiquitination.

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Fig. 4.
Ubiquitination of p53 C-terminal deletion
mutants. A, schematic diagram of human p53. Indicated
are the amino acid boundaries of the transactivation domain (1-42),
DNA binding domain (102-292), oligomerization domain (324-355), and
regulatory domains (367-393). B, Saos-2 cells were
transfected with DNAs encoding wild-type or deleted forms of p53 alone,
or in combination with MDM2, and were examined for p53 ubiquitination
as in Fig. 1. The IgG heavy chain and nonubiquitinated p53
(p53-Ubn) co-migrate at the position of the
asterisk (*). C, p53 and MDM2 protein levels were determined
by immunoblotting without prior immunoprecipitation.
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Coimmunoprecipitation experiments were done to determine the relative
ability of the p53 deletion mutants to bind MDM2 (Fig. 5). High levels of MDM2 were detected in
immunoprecipitates of wild-type p53, p53-(1-363), and p53-(1-353). In
contrast, very low levels of MDM2 coimmunoprecipitated with
p53-(1-333). These results establish a correlation between MDM2
binding and p53 ubiquitination and suggest that deletion of residues
333 to 353 abrogated p53 ubiquitination by diminishing the ability of
p53 to bind MDM2.

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Fig. 5.
MDM2 binding by p53 deletion mutants.
A, Saos-2 cells were transfected with either wild-type p53
or the indicated p53 deletion mutant alone, or in combination with
MDM2. 40 h after transfection, cell lysates were prepared. p53 was
immunoprecipitated (p53 ip) from the lysates with the p53
antibody 1801, and the immunoprecipitates were examined by
immunoblotting with the MDM2 antibody N-20. B, 50 micrograms
of cell extract were examined without prior immunoprecipitation by
immunoblotting with the p53 antibody Ab-6, or the MDM2 antibody
N-20.
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To determine whether oligomerization affected either p53 ubiquitination
or p53·MDM2 binding, the effect of replacing the oligomerization domain of p53 with the dimerization domain from another protein was
determined (Fig. 6). p53-333CC contains
amino acids 1-333 of p53 fused to the dimerization domain from the
yeast transcription factor GCN4 (3). As shown in Fig. 6A,
wild-type p53 and p53-333CC were efficiently ubiquitinated by MDM2,
while p53-(1-333) was not. Further, MDM2 co-immunoprecipitated with
wild-type p53 and p53-333CC to comparable levels but did not
coimmunoprecipitate with p53-(1-333) (Fig. 6B). These
results indicate that a heterologous dimerization domain can restore
MDM2 binding and MDM2-mediated ubiquitination to an
oligomerization-deficient p53. This is consistent with the findings of
Karen Vousden and co-workers (24) who recently reported that p53
proteins with disrupted oligomerization function displayed decreased
MDM2 binding affinity and lower susceptibility to MDM2-mediated
degradation. We conclude that the ability to oligomerize is required
for p53 to efficiently bind MDM2 and be targeted for
ubiquitination.

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Fig. 6.
Dimerization restores MDM2 binding and
ubiquitination to oligomerization-defective p53. Saos-2 cells were
transfected with wild-type p53, p53-(1-333), or p53-333CC singly, or
in combination with MDM2 as indicated. p53-333CC encodes p53 amino
acids 1-333 fused to the GCN4 dimerization domain (3). 20 h after
addition of the DNA precipitate, the transfected cells were split into
two separate dishes. 24 h later, cell extracts were prepared and
examined for p53 ubiquitination (A) or p53·MDM2 complex
formation (B), as described under "Materials and
Methods." In panel A, the asterisk (*) indicates the
position of nonubiquitinated p53 (p53-Ubn) and
the IgG heavy chain used in the immunoprecipitation (ip).
C, 50 micrograms of cell extract were examined without prior
immunoprecipitation by immunoblotting with the p53 antibody Ab-6 or the
MDM2 antibody N-20.
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DNA-damaging agents stabilize p53 through an unknown mechanism. Given
that p53 is targeted for ubiquitination by MDM2, one possibility is
that MDM2-mediated ubiquitination of p53 is inhibited in DNA-damaged
cells. To test this possibility, cells were transfected with p53 alone,
or in combination with MDM2, and subsequently DNA-damaged by exposure
to UV light. The effect of UV exposure on p53 ubiquitination,
p53·MDM2 complex formation, and p53 dimerization was then assessed
(Fig. 7). U2OS cells were used for these
experiments because p53 could be ubiquitinated in these cells in the
absence of MDM2 coexpression. p53 ubiquitination was inhibited in the transfected cells between 1 and 7 h after UV treatment (Fig.
7A). This decrease in ubiquitination coincided with
decreased MDM2 protein levels, and a corresponding decrease in
p53·MDM2 complexes (Fig. 7B). The effect of UV radiation
on p53 dimerization was also assessed. Cells were cotransfected with
DNAs encoding epitope-tagged p53-(HA-p53) and nontagged p53 and
subsequently were exposed to UV radiation. Lysates were
immunoprecipitated with an anti-HA antibody, followed by immunoblot
analysis for p53. The ability of HA-tagged p53 to immunoprecipitate
nontagged p53 was used as a measure of p53 dimerization in
UV-irradiated and nonirradiated cells. As shown in Fig. 7C,
HA-tagged p53 could dimerize nontagged p53 in transfected cells, and
this dimerization was not diminished after UV exposure. These results
indicate that the decrease in p53·MDM2 complex formation following UV
treatment did not involve a decrease in the ability of p53 to
homodimerize.

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Fig. 7.
UV radiation inhibits p53
ubiquitination. A, U2OS cells were transfected with
HA-tagged p53 and exposed to UV radiation (20 J/m2), and
protein extracts were made in RIPA buffer. Upper, p53 was
immunoprecipitated (ip) with the anti-HA polyclonal antibody
HA.11, and the immunoprecipitates were probed with the HA.11 monoclonal
antibody. The ladder of bands are HA-p53:ubiquitin conjugates and were
also recognized by the p53 antibody Ab-6 (not shown).
p53-Ubn, nonubiquitinated p53. Lower,
50 micrograms of protein extract were examined without prior
immunoprecipitation by immunoblotting with the HA.11 monoclonal
antibody. B, U2OS cells were transfected with HA-tagged p53
and were UV irradiated as in panel A, and protein extracts
were made in lysis buffer. Upper, p53 was immunoprecipitated
with the HA.11 polyclonal antibody, and the immunoprecipitates were
probed with the MDM2 antibody N20. Lower, U2OS cells were
transfected with HA-p53 and UV-irradiated as in the upper
panel. Protein extracts were prepared in RIPA buffer 0, 3, or
7 h after UV treatment. p53 levels were assessed at time points
after UV treatment by immunoblotting with Ab-6. MDM2 levels were
assessed by immunoprecipitation with the MDM2 antibody N20, followed by
immunoblotting with N20. C, U2OS cells were transfected with
HA-tagged p53 and nontagged p53 and were UV irradiated as in
panel A. Protein extracts were prepared in lysis buffer, and
p53 was immunoprecipitated with the HA.11 polyclonal antibody. The
immunoprecipitates were probed with the p53 antibody Ab-6. The ability
of HA-tagged p53 to immunoprecipitate nontagged p53 is a measure of p53
dimerization.
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 |
DISCUSSION |
The activity of wild-type p53 is regulated in large part through
changes in protein stability. It is of interest therefore to identify
cellular factors that regulate p53 turnover. The most compelling
evidence that MDM2 normally regulates p53 levels comes from David Lane
and colleagues (25, 26). Phage-display was used in these studies to
identify a 12-amino acid peptide that mimics the MDM2 binding site in
p53 and blocks p53·MDM2 binding. When expressed in cells containing
low p53 levels, this peptide caused an accumulation of p53, activation
of p53-responsive genes, and a cell cycle arrest. In contrast, a mutant
peptide unable to block p53·MDM2 binding had no effect. These results
provided strong evidence that p53 levels and activity are normally
regulated by MDM2 interaction. Other studies demonstrated that MDM2 can promote p53 degradation in transient transfection assays (17, 18)
although the basis of this degradation was not fully clarified. The
current report demonstrates that MDM2 can promote the formation of p53:ubiquitin conjugates in vivo. These results are
consistent with those of Honda et al. (19), which suggested
that MDM2 functions as an E3 ubiquitin protein ligase in p53 ubiquitination.
Cancer-derived p53 mutant proteins varied in their susceptibility to
MDM2-mediated ubiquitination, with the V143A and R248W mutants being
more prone to ubiquitination than the R273H mutant. This did not appear
to result from a lower ability of R273H to bind MDM2, suggesting that
factors other than MDM2 binding can affect p53 ubiquitination. One
possibility is that these p53 mutants vary in their subcellular
localization, and this accounts for their differences in
ubiquitination. It has been suggested that p53 ubiquitination by MDM2
occurs in the nucleus (27), and certain p53 mutants may be retained in
the cytoplasm (28) where they may not be ubiquitinated. The
conformation of p53 might also affect its susceptibility to
ubiquitination. It is worth noting that the V143A and R248W p53 mutants
were more highly ubiquitinated than either wild-type p53 or R273H.
Perhaps R273H adopts a more wild-type conformation than V143A or R248W
and so is less susceptible to ubiquitination.
Deletion analyses indicated that the C-terminal oligomerization domain
of p53 was required for efficient MDM2 binding and ubiquitination. This
is perhaps surprising given that the MDM2 binding site in p53 is
located in the N terminus of p53 (29). Further, MDM2 can interact with
peptides from the N terminus of p53, indicating that C-terminal
sequences are not essential for the interaction to occur. Perhaps the
MDM2 binding domain in p53 assumes a more complex tertiary structure
when p53 is oligomerized, which enhances its affinity for MDM2. These
results are consistent with those of Karen Vousden and her coworkers
(24) who recently demonstrated that p53 proteins with disrupted
oligomerization function displayed decreased binding affinity for MDM2
and were less susceptible to MDM2-mediated degradation.
p53 is stabilized in response to various DNA damaging agents, including
ionizing radiation (5), UV radiation (5), actinomycin D (30), and
cisplatin (31). The molecular basis of this stabilization is unknown.
Two proteolytic pathways have been implicated in p53 degradation; the
calpain proteolysis system (8, 9) and the ubiquitin proteolysis system
(10, 11). Given that p53 is degraded through these two pathways, one
possibility is that DNA damaging agents signal a repression of p53
degradation through the calpain system, the ubiquitin system, or both.
This report demonstrates that p53 ubiquitination is inhibited in
response to UV radiation. This inhibition coincided with decreased
expression of MDM2, and a decrease in p53·MDM2 complexes. This
suggests that UV radiation may stabilize p53, at least in part, by
inhibiting MDM2 expression and thus blocking the ubiquitination and
degradation of p53. DNA-damaging agents can promote p53
phosphorylation, and it has been suggested that this phosphorylation
could stabilize p53 by blocking p53·MDM2 complex formation (16). It
remains possible that such post-translational modifications might also
contribute to the UV-induced loss of p53 ubiquitination observed in the
current report.
Current models suggest that p53 functions in a pathway that monitors
the DNA and protects cells from accumulating genetic damage. p53
carries out this function in response to DNA damage by temporarily
halting cell proliferation to allow DNA repair or by eliminating the
damaged cell through apoptosis (6, 7). This model predicts that p53
inactivation would be necessary for cells to resume cycling following
repair of the damaged DNA. In this regard, it is worth noting the
studies of Perry et al. (32) in which the kinetics of MDM2
and p53 induction were examined in UV-irradiated cells. Three important
findings emerged: 1) the induction of MDM2 was delayed in UV-treated
cells even though p53 levels rose almost immediately; 2) MDM2 induction
was delayed for longer periods of time with increasingly larger UV
doses; and 3) the time of MDM2 induction was correlated with the
recovery of normal rates of DNA synthesis, presumably after DNA repair. Based on these results, it is reasonable to speculate that MDM2 expression may be inhibited in UV-irradiated cells to allow an accumulation of p53 and a subsequent cell cycle arrest and DNA repair.
Once DNA repair is completed, the induction of MDM2 may then allow for
p53 ubiquitination and degradation and for continued cell cycle
progression. It will be of interest to determine whether MDM2 induction
following UV radiation is coincident with repair of the UV-induced DNA
lesions. This would suggest that MDM2-mediated ubiquitination and
degradation of p53 is important for cells to resume cycling after DNA damage.
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FOOTNOTES |
*
This work was supported by a Breast Cancer Research Grant
(to C. M.) from the Massachusetts Dept. of Public Health.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.
To whom correspondence should be addressed. Tel.: 617-432-2532;
Fax: 617-432-2640; E-mail: cmaki{at}hsph.harvard.edu.
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ABBREVIATIONS |
The abbreviations used are:
DMEM, Dulbecco's
modified Eagle's medium;
RIPA, radioimmune precipitation buffer.
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