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Molecular Biology and Genetics |
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong [X. Q. W., A. W. S. L., K. M. L., R. Y. C. P.], and Division of Otolaryngology, Department of Surgery, University of California, San Diego, La Jolla, California 92103 [W. M. O.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The control of MDM2 over p53 is disrupted after DNA damage. After DNA damage, protein kinases like ATM or DNA-PK are activated and phosphorylate p53 at Ser-15 and other sites. These phosphorylations of p53 inhibit the binding of MDM2 to p53, therefore leading to an increase in p53 level, nuclear localization, and transactivation activity (13, 14, 15) . This hypothesis elegantly explains how p53 level and activity are regulated by DNA damage.
MDM2 is overexpressed in a variety of tumors, which would serve to eliminate p53 responses after DNA damage or other stresses (16) . The inhibition of p53 by MDM2 is subjected to regulation by ARF. ARF and the cyclin-dependent kinase inhibitor INK4A are gene products of the INK4A locus on human chromosome 9p21. ARF inhibits the degradation of p53 (17 , 18) by binding to MDM2 and sequestering it into the nucleolus (19, 20, 21) .
Several proteins that share a high degree of sequence similarity to p53 have been identified (22) . These include p73 and its alternatively spliced forms (23, 24, 25) and p63 and its various variants (two of them were also cloned as p51A and p51B; Refs. 26, 27, 28 ). We and others have shown that, like p53, p73 can also bind to MDM2; however, unlike p53, binding of MDM2 to p73 does not target p73 for degradation (29, 30, 31, 32, 33) . Similar to p53, p73 is also induced by certain types of DNA damage (34, 35, 36) . Induction of p73 after DNA damage is dependent on phosphorylation of p73 on Tyr-99 by c-Abl (34, 35, 36) . Interaction between p73 and c-Abl occurs through the SH3 domain of c-Abl and the COOH-terminal homo-oligomerization domain of p73. Recently, it has been shown that E2F-1 activates the transcription of p73 and induces apoptosis (37 , 38) .
Possible physiological functions of the p53-related proteins have been suggested by gene disruption studies in mice. Mice with disrupted p73 have neurological, pheromonal, and inflammatory defects (25) . Unlike p53-/- mice, however, p73-/- mice do not develop tumors spontaneously. One interesting finding about the in vivo function of p73 is that a major form of p73 in the cell is a NH2-terminal-truncated form, which would be incapable of transcriptional activation (25) . The p63 gene product appears to have important functions in development because disruption of p63 in mice affects limb and epithelial development (39 , 40) , and heterozygous germ-line mutations of p63 genes are found in human EEC syndrome (41) .
Here we demonstrate that like p53, p73 could transactivate the MDM2 promoter in p53-null cell lines. The level of MDM2 was increased by p73 because of increases in the transcription and protein stability of MDM2. We demonstrated that the level of p53 could be altered by a cooperation between MDM2 and p73, but not with transcriptional inactive mutants of p73. Expression of p73 resulted in a decrease in exogenous p53 in transient transfection experiments or in endogenous p53 induced by DNA damage. These results suggest the possibility that p53 responses after DNA damage and other stresses can be modulated by different levels of p73.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and p73ß and their transcriptional inactive
mutants (R292H) in pcDNA3 were gifts from Dr. Daniel Caput (Sanofi
Recherche, Labege, France). MDM2 in pCMV was a gift from Dr. Bert
Vogelstein (Howard Hughes Medical Institute, Johns Hopkins Oncology
Center, Baltimore, MD). Human p53 in pRcCMV was a gift from Dr.
Arnold Levine (Princeton University, Princeton, NJ). The
ß-galactosidase construct was a gift from Dr. Yan Chen (Indiana
University School of Medicine, IN). CD2 construct was a gift
from Dr. Chris Norbury (ICRF, University of Oxford, Oxford, United
Kingdom). MDM2 promoter-luciferase reporter construct was a gift from
Dr. Moshe Oren (The Weizmann Institute, Rehovot, Israel).
Cell Culture and Transfection.
H1299 cells (human non-small cell lung carcinoma), MCF-7 (human mammary
adenocarcinoma), SAOS-2 (human osteogenic sarcoma), and U2OS (human
osteosarcoma) were obtained from the American Type Culture Collection
(Manassas, VA). Cells were grown in DMEM supplemented with 10% v/v
fetal bovine serum in a humidified incubator at 37°C with 5%
CO2. Semiconfluent cells were transiently
transfected with the calcium phosphate precipitation method
(42)
. Unless stated otherwise, 10 µg of each plasmid
were used to transfect cells in 10-cm plates. The total amount of DNA
for each transfection was adjusted to the same level using vectors with
the same promoter. Cells were grown for an additional 24 h after
transfection before harvesting cell extracts. Cell-free extracts were
prepared as described previously (43)
. The protein
concentration of cell lysates was measured with the bicinchoninic acid
protein assay system (Pierce) using BSA as a standard. Selection of CD2
cell surface marker was performed according to the manufacturer’s
instructions (DYNAL, Oslo, Norway).
Luciferase and ß-Galactosidase Assays.
Luciferase assays and ß-galactosidase assays were performed exactly
as described previously (44)
.
Antibodies and Immunological Methods.
Monoclonal antibody DO-1 against p53 (sc-126) and rabbit
anti-p21CIP1/WAF1 antibodies (sc397) were from
Santa Cruz Biotechnology. Monoclonal antibody 2A10 against MDM2 was a
gift from Dr. Arnold Levine. Ab-1 against MDM2 was from Oncogene
Science, and 12CA5 against the HA tag was from Roche Molecular
Biochemicals. Anti-CD2 (OX-34) conjugated to magnetic beads (DYNAL)
was a gift from Dr. Chris Norbury; IF-6 antibody against
topoisomerase II was a gift from Dr. Ian Hickson (ICRF, University
of Oxford); and YL1/2 against tubulin was a gift from Dr. Tim Hunt
(ICRF, South Mimms, United Kingdom). Immunoblottings
(45)
and immunoprecipitations (46)
were
performed as described previously. When indicated, signals on
immunoblots were analyzed using the NIH Image program with the
appropriate serial dilution standard curves.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cotransfection of the same amount of MDM2 plasmid
inhibited the transcriptional activity of p53 by about 80%.
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shows that like p53, expression of p73 in H1299
cells transactivated the MDM2 promoter. In contrast to p53,
cotransfection of the same amount of MDM2 with p73 did not result in
an inhibition of the transcriptional activity of p73. As a control,
a transcriptional inactive mutant of p73 (R292H) was not able to
transactivate the MDM2 promoter and was also not affected by MDM2.
Similar results were seen with the alternatively spliced p73ß, as
well as in another p53-null cell line, SAOS-2 (data not shown). We
found that transactivation of the MDM2 promoter by p73 was inhibited
when the amount of MDM2-expressing plasmid was progressively raised
(Fig. 1C)
, suggesting that the transcriptional activity of
p73 could be inactivated by a higher concentration of MDM2. These data
indicate that the MDM2 promoter can be transactivated by p73 in a
p53-null background. Furthermore, inhibition of p73 appears to require
a higher concentration of MDM2 than inhibition of p53, although
both p73 and p53 were driven by similar CMV promoters. One explanation
is from our previous data, which show that MDM2 binds to p73 but does
not target p73 for degradation (31)
. Hence, at a low
concentration of MDM2, p53 but not p73 is destabilized. However, at a
high concentration of MDM2, both p53 and p73 are saturated with MDM2,
and their transcriptional activities are inhibited.
We next studied whether the protein level of MDM2 is affected by
expression of p73. Accumulation of endogenous MDM2 (both the
Mr 55,000 and the
Mr 90,000 forms) was observed when
p73 or p73ß was overexpressed in SAOS-2 or H1299 cells (Fig. 2
, Lanes 2 and 5). It is interesting that after
transfection of p73, both the Mr
55,000 and Mr 90,000 forms of
endogenous MDM2 were seen in SAOS-2 cells (Lane 2), but only
the Mr 90,000 form was present in
H1299 cells (Lane 5). As we have shown previously
(31)
, coexpression of p73 with MDM2 resulted in the
stabilization of p73 (Lanes 3 and 6). A similar
effect of p73 on endogenous MDM2 could be seen with the p53-positive
U2OS cells (see Fig. 6C
). Importantly, the R292H
transcriptional inactive mutant of p73 caused a substantially
smaller increase in MDM2 (Fig. 6C)
, suggesting that
transcriptional activity of p73 was required for the increase in MDM2.
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shows that MDM2 protein was significantly more stable in cells that
were cotransfected with p73 plasmids than in cells transfected with
MDM2 plasmids alone. These data do not necessarily imply that a direct
interaction between p73 and MDM2 was responsible for the stabilization
of MDM2. It is conceivable that cell cycle arrest or other effects
caused by p73 have an indirect effect on the MDM2 stability. Taken
together, these data indicate that expression of p73 could lead to an
increase in MDM2, possibly due to increases in both the transcription
and protein stability of MDM2.
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, Lanes 1–3). Significantly,
in the presence of p73 or p73ß, p53 expression was further reduced
(Lanes 4 and 5). A similar amount of total cell
lysates was loaded in each lane as indicated by immunoblotting for
tubulin. Interestingly, the level of transfected MDM2 was not
significantly increased by the presence of p73 (although the amount of
endogenous MDM2 was increased by p73; see Fig. 2
). This suggests
that the destabilization of p53 by p73 could be due to a mechanism
other than the transfected MDM2.
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, Lanes 1 and 3). Strikingly,
expression of p73 in these cells reduced the level of p53 in untreated
cells (Lanes 1 and 2). Moreover, expression of
p73 also abolished the induction of p53 by Adriamycin (Lane
4). Similarly, we found that p53 could be induced in U2OS cells by
UV in a time-dependent manner (Fig. 5B)
. The induction of
p53 by UV was delayed when p73 was expressed in the cells (Lanes
3 and 4). A more profound inhibition of p53 responses
to Adriamycin and UV by p73 was seen in the human mammary
adenocarcinoma MCF-7 cells (Fig. 5, C and D)
. The
levels of tubulin or topoisomerase II were used to indicate similar
loading of total cell lysates in the above-mentioned experiments.
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shows that coexpression of p73 decreased the amount of p53
found in the MDM2 immunoprecipitates (Lanes 1 and
2). The same results were obtained with endogenous p53 and
MDM2 in U2OS cells in the absence (Fig. 6A
, Lanes
3 and 4) or presence (Fig. 6B)
of DNA
damage. In these U2OS cells, endogenous MDM2 was strongly induced when
p73 was expressed (Fig. 6
C, Lane 2). A substantially lower
induction of MDM2 protein was seen when the transcriptional inactive
mutant of p73 (R292H) was expressed (Lane 3). Hence the
decrease in MDM2-p53 complexes occurred when the MDM2 level was
increased. One possibility is that the MDM2 that was induced by p73
expression could bind to p53 and target p53 for degradation. This
resulted in a reduction of the total amount of p53 and of the
p53 that associated with MDM2 at the end of the experiment.
To test the above-mentioned hypothesis, we used the proteasome
inhibitor LLnL to block the degradation of p53. Fig. 6D
shows that expression of p73 but not R292H mutant
decreased the level of p53 that associated with MDM2 (Lanes
2–4). This datum shows that the reduction of p53 level by p73
required the transcriptional activity of p73. Treatment with LLnL
increased the amount of p53 associated with MDM2 (Lanes
5–7). Furthermore, a higher ratio of increase of p53 after LLnL
treatment was seen in cells expressing p73 in comparison with cells
transfected with control vector or p73 (R292H) mutant. We also
consistently found that in the presence of LLnL, there was slightly
more p53 associated with MDM2 in p73-transfected cells than in cells
transfected with control vector or p73 (R292H) mutant. Taken together,
these data indicate that expression of p73 can modulate the level of
p53. This modulation of p53 is likely to be due to an increase in MDM2
level (by transcription and protein stabilization), which in turn can
target p53 for degradation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and p73ß (Fig. 1)
. The transcriptional
targets of p73 are similar but not identical to those of p53
(22)
. The MDM2 promoter can be activated to varying
degrees by different isoforms of p73 (29
, 33
, 47, 48, 49)
,
although other studies showed no effect of p73 on MDM2 transcription
(50)
. Furthermore, the activation of the
p21CIP1/WAF1 promoter by p73 appears to be
different from p53. We think that one of the major reasons that p53
decreased when p73 was expressed is because MDM2 was transactivated by
p73, which in turn interacted with p53 and targeted it for degradation. Conceptually, the MDM2 that is induced by p73 can bind to either p53 or p73. Binding of MDM2 to p53 targets p53 for degradation, but binding of MDM2 to p73 stabilizes p73 (52) . In support of this, there is an increase in MDM2-p73 complex formation when MDM2 is coexpressed with p73 (31) . The molecular basis that underlies p73 binding to MDM2 without subsequent degradation is unknown. One possibility is that the COOH terminus of p53, which is necessary for efficient MDM2-mediated degradation of p53, is not conserved in p73 (53) . Recently, Gu et al. (54) have defined the sequence element unique to p53 that is absent in p73 (residues 92–112 of p53), which is essential for its regulation by MDM2.
In contrast to p73, p53 is rapidly degraded when it interacts with
MDM2. Hence, relatively little p53 could be detected in the MDM2
immunoprecipitates unless it is stabilized by the proteasome inhibitor
LLnL (Fig. 6)
. Given that both p53 and p73 could bind to MDM2, it is
possible that p53 and p73 could compete for MDM2. This would tend to
produce an increasing amount of p73-MDM2 complexes and a decreasing
amount of p53-MDM2 complexes. The fact that expression of p73 resulted
in a reduction of p53 indicates that MDM2 was available for binding to
p53 even when p73 was overexpressed. This could be because the affinity
of MDM2 for p53 is higher than that for p73 or because the level of
MDM2 is higher than that of p73.
It is interesting that MDM2 protein was stabilized in the presence of
p73 (Fig. 3)
. MDM2 is degraded by the ubiquitination proteasome
pathway, but there is no report that MDM2 can be stabilized by binding
to p73. As discussed above, our data do not necessarily imply that a
direct interaction between p73 and MDM2 was responsible for the
stabilization of MDM2. It has been shown that binding of MDM2 to other
proteins, such as the MDM2-related protein MDMX, can stabilize MDM2
(55
, 56)
.
p73 has been reported to interact with p53 in other respects.
Hetero-oligomers between the p53 and p73 family are possible, but they
tend to form homo-oligomers (22)
. In another report, when
p73 was expressed in human ovarian carcinoma cell line A2780,
endogenous p53 transcriptional activity was markedly decreased
(57)
. Vikhanskaya et al. (57)
attributed this to a possible sequestration of p53 from its DNA binding
site through competitive binding with p73. Conversely, it is
conceivable that the expression level of p53 could also affect the
level and activity of p73. In this connection, it has been shown that
some tumor-derived mutants of p53 can inhibit the function of p73
(49)
. The mechanisms of this inhibition are unknown.
Whether the stabilization of p73 by MDM2 produces more active p73 or
more inactive MDM2-p73 complexes is still a contentious issue. MDM2 is
able to reduce p73-dependent transcription in some in vitro
reporter assays (30
, 32
, 33)
, but the growth-inhibitory
activity of p73 is increased in the presence of MDM2 (31)
.
It is conceivable that there is a balance between the increase in p73
transcription activity due to an increase in protein stability and the
decrease in p73 transcription activity due to binding of MDM2 to the
transactivation domain. In support of this argument, we found that a
relatively high concentration of MDM2 was required to inhibit
p73-dependent transcription of the MDM2 promoter (Fig. 1)
. It is likely
that the effect of MDM2 on p73 may be different with different
p73-responsive promoters. For instance, we found that coexpression with
MDM2 significantly increased p73 transactivation activity on the
p21CIP1/WAF1
promoter.5
An important inference from these data is that we expect that an increase in p73 (rather than a loss of function as in p53) can lead to deregulation of the DNA damage checkpoint. This is due to an indirect effect because elevated levels of p73 can lead to a reduction of active p53. This may explain the fact that very little loss of function of p73 has been seen in cancer and that p73-deficient mice do not develop tumors spontaneously (25) . Hence, it would be very interesting to examine whether p73 is overexpressed in any cancer tissues. Another prediction from the hypothesis presented here is that the level of p53 may decrease when p73 is stabilized after the activation of c-Abl.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Research Grants Council
Grants HKUST6090/98 M and DAG99/00.SC03 (to R. Y. C. P.). 
2 Present address: Department of Radiation
Oncology, Med. Sci. I B140, University of California, Irvine, CA
92697.
3 To whom requests for reprints should be
addressed, at Department of Biochemistry, Hong Kong University of
Science and Technology, Clear Water Bay, Hong Kong. Phone:
852-2358-8703; Fax: 852-2358-1552; E-mail: bcrandy{at}ust.hk
4 The abbreviations used are: HA, hemagglutinin;
ICRF, Imperial Cancer Research Fund; CMV, cytomegalovirus.
Received 3/28/00. Accepted 12/13/00.
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and 
, with different transcriptional activity.. J. Exp. Med., 188: 1763-1768, 1998. without targeting degradation.. Oncogene, 18: 3923-3929, 1999.[Medline]
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