Oncogene (1999) 18, 7656 7665

1999 Stockton Press All rights reserved 0950 9232/99 $15.00

http://www.stockton-press.co.uk/onc

p53 regulation by post-translational modication and nuclear retention in

response to diverse stresses
Gretchen S Jimenez1, Shireen H Khan1,2, Jayne M Stommel1,2 and Georey M Wahl*,1
1
2

Gene Expression Laboratory, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, California, CA 92037, USA;
Department of Biology, University of California at San Diego, La Jolla, California, CA 92037, USA

p53 activation by diverse stresses involves post-translational modications that alter its structure and result in
its nuclear accumulation. We will discuss several
unresolved topics regarding p53 regulation which are
currently under investigation. DNA damage is perhaps
the best-studied stress which activates p53, and recent
data implicate phosphorylation at N-terminal serine
residues as critical in this process. We discuss recent
data regarding the potential kinases which modify p53
and the possible role of the resulting phosphorylation
events. By contrast, much less is understood about agents
which disrupt the mitotic spindle. The cell cycle phase,
induction signal, and biochemical mechanism of the
reversible arrest induced by microtubule disruption are
currently under investigation. Finally, a key event in
response to any genotoxic stress is the accumulation of
p53 in the nucleus. The factors which determine the
steady state level of p53 are starting to be elucidated, but
the mechanisms responsible for nuclear accumulation and
nuclear export remain controversial. We discuss new
studies revealing a mechanism for nuclear retention of
p53, and the potential contributions of MDM2 to this
process.
Keywords: p53; DNA damage; nuclear export signal;
microtubule depolymerization; post translational modication

Introduction
New revelations continue to emerge concerning the
mechanisms that control p53 activation in response to
a wide range of input signals. This tumor suppressor
maintains genetic stability by responding to multiple
environmental stresses including DNA damage, ribonucleotide depletion, microtubule disruption, redox
modulation, cell adhesion, and hypoxia, as well as
`genetic' stresses created by activated oncogenes
(reviewed in Giaccia and Kastan, 1998). These diverse
stimuli appear to invoke a similar set of responses to
achieve p53 activation: p53 must rst accumulate in the
nucleus, and then bind to DNA as a tetramer to
transcriptionally regulate a growing list of target genes
including p21, GADD45, cyclin G, 14-3-3sigma,
thrombospondin 1, MDM2, IGF-BP3, and bax
(reviewed in El-Deiry, 1998; Gottlieb and Oren, 1996;
Ko and Prives, 1996), as well as c-fos (Elkeles et al.,
1999) and others. However, depending on the stress
and cell type, p53 activation can lead to responses as
*Correspondence: GM Wahl

dierent as a reversible cycle arrest, induction of a

senescent-like state, or apoptosis. Clearly, the induction
of the irreversible states of senescence or apoptosis
requires a permanent activation of p53 which commits
the cell to exit the cycle forever. On the other hand,
some agents, such as nocodazole and colcemid, cause a
reversible arrest. This raises the important question of
whether stimuli that induce cell cycle exit induce
dierent alterations in p53 than those that result in a
reversible response, which could result in regulation of
dierent downstream genes. We infer that there must
also be attenuation mechanisms that reverse p53
function subsequent to removal of certain stresses.
Since p53 has the power to either target a cell for
death or allow it to survive, it must be under rigorous
and complex control. p53 contains an N-terminal
transactivation domain, a core DNA binding domain,
and a C-terminal multifunctional regulatory domain
(reviewed in Ko and Prives, 1996). Highly conserved
residues in its N- and C-terminal domains are targets
for potential post-translational modication via phosphorylation, dephosphorylation or acetylation (reviewed in Giaccia and Kastan, 1998). We will discuss
how post-translational modication may inuence p53
function in a variety of ways. The possibilities include
eects on binding to other proteins, such as coactivators or negative regulators, and induction of
higher order structural changes mediated by highly
conserved amino acid motifs encoded within the
primary sequence. For example the multifunctional
C-terminus contains a newly identied nuclear export
signal (NES) within the tetramerization domain
(Stommel et al., 1999). Data are discussed which
indicate that the NES is occluded in the p53
tetramer, the active DNA binding conformation.
N-terminal phosphorylation and p53 activation
p53 protein is translocated to the nucleus, stabilized
and rendered competent for DNA binding and
transactivation in response to DNA damage. Many
studies have attempted to correlate the contributions of
p53 protein accumulation, post-translational modification, and binding to regulatory proteins with the ability
of p53 to bind DNA and transactivate downstream
target genes. p53 transactivation appears to require its
interaction with components of TFIID, including the
TATA box binding protein (TBP) and TBP-associated
factors (TAFs), via the p53 N-terminal transactivation
domain (Lu and Levine, 1995; Thut et al., 1995; Xiao
et al., 1994). More recently, transcriptional coactivators p300 and CREB-binding protein (CBP)
have also been shown to enhance p53 transactivation

Regulation of p53 by diverse stresses

GS Jimenez et al

(Gu et al., 1997; Lill et al., 1997; Scolnick et al., 1997).

On the other hand, binding of MDM2, which is a
downstream p53 target, downregulates p53 transactivation (Wu et al., 1993). This may be explained by both
steric interference between MDM2 and the coactivators, as MDM2 binds p53 at amino acids 17
27 (Chen et al., 1993; Kussie et al., 1996). As MDM2
also targets p53 for degradation (Haupt et al., 1997;
Kubbutat et al., 1997, 1998), this region is crucial to
the concurrent regulation of p53 transactivation and
stability.
Phosphorylation of N-terminal amino acids could
contribute to p53 regulation by aecting the binding of
co-activators and the negative regulator MDM2. p53 is
a good in vitro kinase substrate for casein kinase I
(CKI) at S6 and S9 (Milne et al., 1992), DNAdependent protein kinase (DNA-PK) at S15 and S37
(Lees-Miller et al., 1992; Shieh et al., 1997), c-Jun
kinase (JNK) at S37 (Milne et al., 1995), and CDK
activating kinase (CAK) at S33 (Ko et al., 1997). The
importance of these potential phosphorylation sites has
been assessed by mutating them individually or in
combination and (over)expressing the respective
mutants in various cell lines (Ashcroft et al., 1999;
Fiscella et al., 1993; Fuchs et al., 1995; Mayr et al.,
1995). These studies have yielded conicting results,
but indicated an importance for phosphorylation at
S15 and S37 in maintaining protein stability and
transactivation properties. The importance of Nterminal phosphorylation was assessed in vivo in
recent experiments using two dimensional peptide
mapping to show that in response to ionizing
radiation (IR), three sites in the N-terminus of p53
are phosphorylated, two of which reside in the rst 24
amino acids (Siliciano et al., 1997). One interpretation
consistent with these data is that N-terminal phosphorylation interferes with MDM2 binding, which would be
expected to allow association of p53 with co-activators
and prevent MDM2 from targeting p53 for cytoplasmic degradation (Pise-Masison et al., 1998; Shieh et al.,
1997). Phosphorylation of p53 at S15 and S37 in vitro
with puried DNA dependent protein kinase (DNAPK) impaired not only the ability of MDM2 to interact
with p53, but also its ability to block p53-dependent
transcription (Shieh et al., 1997). This model was
further supported in vivo by a damage-induced decrease
in the binding of MDM2 to p53. In addition,
immunoprecipitation studies using an antibody specic for phosphoserine 15 showed that this site is
phosphorylated in response to DNA damage (Siliciano et al., 1997).
The contribution of N-terminal phosphorylation to
the damage response is becoming increasingly clear,
but the identication of the upstream kinases which
respond to various stresses is still being debated. Three
members of the PIK-related kinase family, DNA-PK,
ATM (the kinase mutated in patients with ataxia
telangiectasia), and ATR (the ataxia telangiectasia
related kinase), have been proposed as upstream
activators of p53 (Banin et al., 1998; Canman et al.,
1998; Kastan et al., 1992; Shieh et al., 1997; Siliciano et
al., 1997; Tibbetts et al., 1999; Woo et al., 1998).
DNA-PK could easily be activated by DNA damage as
it is recruited to diverse aberrant DNA structures by
the Ku70/80 heterodimer which binds the DNA-PK
catalytic subunit (cs) (reviewed in Jeggo, 1997; Smith

and Jackson, 1999). DNA-PK subunits are suciently

abundant to locate, bind to, and become activated by a
solitary double strand break, which is the minimum
amount of damage needed to trigger a p53-dependent
arrest response (Di Leonardo et al., 1994; Huang et al.,
1996).
Even though DNA-PK phosphorylates p53 in vitro
at S15 and S37, a signicant controversy has emerged
over whether it plays a physiological role in p53
activation (Lees-Miller et al., 1992). Until recently,
studies involving DNA-PK utilized cells derived from
mice with a form of severe combined immunodeciency
(SCID) (Blunt et al., 1995; Kirchgessner et al., 1995).
Primary embryonic broblasts from SCID mice
arrested like wildtype cells in response to DNA
damage (Fried et al., 1996; Guidos et al., 1996;
Gurley and Kemp, 1996; Huang et al., 1996; Nacht
et al., 1996; Rathmell et al., 1997), which is not
expected if DNA-PK is required to generate the Nterminal phosphorylations that prevent MDM2 binding. However, the SCID MEFs may have sucient
residual kinase activity for p53 to be activated with
near normal kinetics (Danska et al., 1996; Woo et al.,
1998). Data suggesting the importance of DNA-PK in
p53 activation emerged from a recent study utilizing
SCGR11, a transformed cell line derived from SCID
mouse broblasts. p53-dependent DNA binding and
transactivation were shown to be aberrant in these
cells. The largely in vitro studies led the authors to
conclude that DNA-PK is necessary, but not sucient,
for induction of p53 DNA binding and transcriptional
activity (Woo et al., 1998).
We used primary mouse embryonic broblasts
(MEFs) derived from mice in which the DNA-PK
gene was inactivated via homologous recombination in
order to compare the cell cycle responses and p53
activation kinetics to those in the SCGR11 cell line.
We showed that SCGR11 contains a mutation in the
p53 core DNA binding domain (Jimenez et al., 1999).
This mutation is similar to those found in many
cancers (Hainaut and Milner, 1993), which explains the
inability of p53 target genes to be induced in SCGR11
cells and the lack of DNA binding exhibited by
SCGR11 derived p53. During our rigorous analysis
of each step involved in a p53 dependent response
utilizing DNA-PKcs knockout MEFs, we showed that
in response to DNA damage, p53 accumulates in the
nucleus, is phosphorylated on the mouse equivalent of
S15, binds DNA, transactivates target genes, and
mediates a robust G1 phase cell cycle arrest with
kinetics which are similar to those of normal cells
(Jimenez et al., 1999). Therefore, DNA-PK activity is
not required to activate p53 in response to DNA
damage.
The ATM and ATR kinases are much stronger
candidates for upstream regulators of p53. AT decient
cells exhibit a delayed p53 accumulation after ionizing
radiation (IR) (Canman et al., 1994; Kastan et al.,
1992; Lavin et al., 1994; Zu and Baltimore, 1996) with
an accompanying delay in S15 phosphorylation
(Siliciano et al., 1997). In vivo and in vitro evidence
shows that ATM and ATR will phosphorylate p53 on
S15 (Banin et al., 1998; Canman et al., 1998; Khanna
et al., 1998; Tibbetts et al., 1999). Although the
response is delayed, p53 is eventually induced and
phosphorylated in IR-treated AT decient cells

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7658

(Siliciano et al., 1997). This suggests that another

kinase, such as ATR, may take over for some of the
ATM function in cells obtained from AT patients or
atm7/7 mice. In fact, overexpression of an inactive
form of ATR in normal broblasts mimics several
phenotypes of AT defective cells (Cliby et al., 1998).
Interestingly, p53 protein accumulation and S15
phosphorylation occur with normal kinetics in UVirradiated AT decient cells (Canman et al., 1994;
Khanna and Lavin, 1993; Siliciano et al., 1997). Since
the UV response is normal in AT decient cells, it
appears that dierent upstream signal transducers
activate p53 in response to dierent forms of DNA
damage.
While many studies have focused on the N-terminal
modication events in response to ionizing or gamma
radiation, the phosphorylation of S15 and S37 are
likely to represent only a fraction of the modications.
For example, recent data suggest that phosphorylation
of S20, not S15, disrupts the association of MDM2
with p53 (Chabib et al., submitted; Shieh et al., 1999;
Unger et al., 1999). Therefore, S20 could be the second
damage-responsive phosphorylation site in the rst 24
amino acids indicated by 2 D peptide mapping
(Siliciano et al., 1997). The identity of the kinase
responsible for this modication has not been
determined.
The question remains as to the importance of
phosphorylation in maintaining protein stability
versus activation of p53 as a transcription factor. For
example, unmodied p53, which has been stabilized by
treatment with a proteasome inhibitor, shows the same
kinetics of binding to and dissociation from DNA as
p53 from IR-treated cells, as monitored by electrophoretic mobility shift assay (Siliciano et al., 1997).
However, p53 in cells treated with proteasome inhibitor
does not have the same ability to transactivate
downstream targets as the p53 in IR-treated cells
(Siliciano et al., 1997). Although these data indicate
that post-translational modication is important, p53
retained transciptional transactivation function after
mutation of all known phosphorylation sites in the Nand C-termini to alanine (Ashcroft et al., 1999). Only
mutation of S15 and S37 to aspartic acid disrupted
MDM2-mediated degradation, but only slightly
(Ashcroft et al., 1999).
The C-terminus contains several sites of potential
modication, and interactions of the C-terminus with
residues in the middle of the protein have long been
known to prevent p53 from binding DNA (Hupp et al.,
1992; Ko and Prives, 1996). p53 is phosphorylated in
vitro on S315 by cdc2 and cdk2, on S378 by protein
kinase C, and on S392 by casein kinase II (Milczarek et
al., 1997). There is also growing evidence for activation
of p53 through dephosphorylation. For instance, S376
appears phosphorylated in unirradiated cells but
becomes dephosphorylated rapidly subsequent to
ionizing irradiation (Waterman et al., 1998). This
dephosphorylation event leads to association with a
14-3-3 protein, which correlates temporally with p53
activation (Waterman et al., 1998). Similarly, acetylation of the C-terminus on K382 by p300 or on K320 by
PCAF enhances sequence specic DNA binding in vitro
(Liu et al., 1999; Sakaguchi et al., 1998). Acetylationspecic and phosphospecic antibodies reveal that S33
and S37 are phosphorylated after UV or IR, along

with acetylation at K382, while K320 is acetylated only

after UV (Sakaguchi et al., 1998). In vitro studies
indicate that the N-terminal phosphorylation events
may facilitate C-terminal acetylation (Sakaguchi et al.,
1998). More recently, it has been reported that the
tetramerization domain is necessary to facilitate Nterminal p53 phosphorylation (Shieh et al., 1999).
Therefore, multiple modications on both ends of p53,
coupled with the structural changes they elicit, are
likely to occur in response to DNA damage. These
modications appear to inuence both the association
of p53 with regulatory proteins and its ability to
regulate the transcription of target genes.
Despite the correlations between damage and p53
modication, there is still much confusion over the role
of post-translational modication in p53 function. We
discussed above the potential delicate interplay of
several modications of p53 in mediating a damage
response, raising the expectation that their mutation
would lead to gross alterations in p53 function.
Conicting data regarding the contribution of individual phosphorylation events to p53 function suggest
that either the contribution of post-translational
modication is subtle, or that experimental systems
which cannot accurately recapitulate p53 expression
may fail to reveal the eects of such mutations on cell
cycle progression, apoptosis, genetic instability and
other important biological manifestations of p53
function. With the growing number of specialized
antibodies recognizing the acetylated or phosphorylated forms of p53, study of the events occurring in vivo
during expression of the endogenous gene should
become practical. Another important target for future
study is the modication of p53-associated proteins.
For instance, it was shown that DNA-PK could
phosphorylate MDM2 in vitro (Mayo et al., 1997). It
may be that analysis of both p53 and its binding
partners will be required to understand which
modications on each protein play important biological roles. Finally, new elements intrinsic to the p53
protein itself, such as the newly identied nuclear
export signal (NES) within the tetramerization domain,
point us toward new levels of regulation in p53
function which are only now being elucidated (see
below).
Disruption of microtubule organization induces an arrest
in a 4N G1-like state via the components of the p53 G1
arrest pathway
Faithful genome replication and maintenance of
diploidy are commonly compromised in human
neoplasms. The components of the machinery that
ensure that duplicated chromosomes are segregated
evenly between two daughter cells remain to be
elucidated. However, recent data implicate p53 as
part of a checkpoint that senses microtubule disruption, and that prevents the outgrowth of aneuploid
variants that emerge after exposure of cells to
microtubule inhibitors.
A key link was established when Brian Reid and
colleagues demonstrated that in rodent cells microtubule disruption in combination with p53 loss allows
downstream events (DNA replication) to occur prior to
the successful completion of upstream events (mitosis)

Regulation of p53 by diverse stresses

GS Jimenez et al

(Cross et al., 1995). The consequence of this loss of

coordination is an increase in the number of cells with
greater than 4N DNA content. Thus, loss of p53 leads
to DNA reduplication after exposure to agents that
inhibit mitotic division, such as nocodazole and
colcemid. These data led to the proposal that p53
functions in a mitotic spindle checkpoint in which it
monitors the proper assembly of the mitotic spindle
and prevents exit from mitosis under conditions that
hinder cell division (Cross et al., 1995). Subsequent
studies showed that loss of pRb function in rodent
broblasts led to an increase in polyploidy after
exposure to nocodazole or colcemid for extended time
periods (Di Leonardo et al., 1997a). Hence, loss of p53
and pRb allow DNA replication to occur prior to
completion of the upstream mitotic events.
Rereplication was also observed in human broblasts
lacking functional p53 or pRb due to inactivation by
HPV16 E6 or E7, suggesting that the controls
coordinating the proper progression of the cycle are
conserved between species (Di Leonardo et al., 1997b;
Thomas et al., 1996). However, others report that DNA
reduplication only occurs in human broblasts expressing p53 missense mutations which confer a dominant
gain-of-function phenotype (Gualberto et al., 1998). The
authors argue that loss of p53 is not sucient to induce
polyploidization in human cells, but rather, a specic
class of p53 missense mutations imparts an oncogenic
phenotype that promotes polyploidy. The conicting
reports may reect dierences in cell types, drug
concentrations and length of exposure to the microtubule destabilizing agents. In fact, high concentrations
of nocodazole were shown to prevent rereplication in
baby hamster kidney cells (Andreassen and Margolis,
1994). Moreover, several dierent cell lines varied in
their propensity to rereplicate after colcemid treatment,
suggesting that cell types dier in their response to
microtubule inhibitors (Kung et al., 1990).
The available data allow two possible models to be
proposed, one of which implicates p53 and pRb in
novel roles during G2/M to prevent cell cycle
advancement in the presence of spindle poisons.
This would be analogous to the Saccharomyces
cerevisiae bub mutants that become polyploid under
microtubule depolymerizing conditions (Hoyt et al.,
1991). BUB1 encodes a protein kinase that is
required to arrest cells prior to anaphase following
spindle disruption (Farr and Hoyt, 1998; Roberts et
al., 1994). A second model proposes that extended
exposure to microtubule destabilizing agents does not
result in a long term arrest in mitosis; rather, the
cells escape the mitotic block and enter a stage
similar to G1 during which p53 and pRB mediate the
arrest response. Persuasive data from several dierent
experimental approaches demonstrate that extended
exposure to microtubule destabilizing agents does not
lead to a sustained mitotic arrest in mammalian cells.
Instead, the cells undergo `mitotic slippage' whereby
cells with a 4N DNA content bypass the mitotic
block and maintain a long term arrest in a stage
biochemically similar to G1 (Andreassen and
Margolis, 1994; Kung et al., 1990; Rieder and
Palazzo, 1992). This raises the possibility that p53
and pRb prevent reduplication after the cells have
undergone mitotic slippage and enter a 4N `G1-like'
state. We will refer to this stage as a `G1-like' stage

since the classical denition of G1 requires that the

cells have 2N DNA content.
There is now compelling evidence that, independent
of the p53 or pRb status, extended exposure of
mammalian broblasts to nocodazole leads to a
transient mitotic arrest during which the cells have
elevated levels of cyclin B1, MPM-2 reactivity (a
marker of M phase), condensed chromosomes, and a
rounded mitotic morphology (Bunz et al., 1998; Khan
and Wahl, 1998; Lanni and Jacks, 1998; Minn et al.,
1996; Notterman et al., 1998). The cells may spend up
to 6 h struggling through mitosis before adapting to
the presence of nocodazole and escaping the mitotic
block (Lanni and Jacks, 1998). Once the broblasts
bypass this block, they enter a phase exhibiting many
characteristics of G1: cyclin B1 levels are low, cyclins
D and E are high, p53 activation occurs, and pRb is
hypophosphorylated (Khan and Wahl, 1998; Lanni
and Jacks, 1998; Minn et al., 1996; Notterman et al.,
1998). The nocodazole induced rereplication observed
in p53 and pRb-decient cells occurs only after a
signcant delay, which corresponds to the length of
time required for the fastest progressing cells to escape
the mitotic block. Taken together, the data support a
role for p53 and pRb in preventing entry into the
ensuing S phase by mediating an arrest during a G1like state.
p53 mediates a G1 arrest in response to DNA
damage and ribonucleotide depletion, in part, by
activating the cyclin dependent kinase inhibitor p21
(El-Deiry et al., 1993; Harper et al., 1993). Mouse
embryonic broblasts lacking p21 were originally
reported to arrest after microtubule disruption (Deng
et al., 1995); however, subsequent reports showed that
p217/7 MEFs rereplicate after exposure to nocodazole or colcemid and induction of the p21 protein
occurs within 24 h in wildtype, but not p53 null MEFs
(Khan and Wahl, 1998; Lanni and Jacks, 1998). This
suggests that p53 mediates the nocodazole induced G1
arrest, in part, through activation of p21. Curiously,
overexpression of a transactivation-decient form of
p53 in immortal murine broblasts apparently can also
prevent rereplication upon exposure to microtubule
inhibitors, suggesting that the p53-mediated activation
of p21 is not absolutely required to induce the arrest
response (Notterman et al., 1998). Indeed, a loss of p53
leads to a greater proportion of polyploid cells than
does p21 loss (Khan and Wahl, 1998), it is possible
that the role of p53 in this response transcends p21
activation. However, it is also possible that overexpression of certain mutant forms of p53 may
produce the observed microtubule inhibitor-induced
cell cycle arrest by interfering with gene expression in
ways that would not occur with normal levels of p53
expression. It remains to be determined if endogenous
levels of transcriptionally defective mutant p53 also
retain the ability to prevent rereplication in the
presence of nocodazole.
Mouse embryonic broblasts lacking both p16Ink4a
and p19ARF also showed increased polyploidy after
exposure to nocodazole or colcemid, suggesting that
the loss of pRb and/or p53 regulation in these MEFs
promotes DNA reduplication (Khan and Wahl, 1998).
This raises questions about the individual roles of the
tumor suppressors p16Ink4a and p19ARF in preventing rereplication. p16Ink4a may prevent S phase entry

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GS Jimenez et al

7660

by inhibiting the phosphorylation of pRb which

promotes the sequestration of the E2F family of
transcription factors (Chellappan et al., 1991; Serrano
et al., 1993). p19ARF may also promote G1 arrest in
the presence of microtubule antagonists by interfering
with the MDM2-mediated degradation of p53 and
thereby promoting the stability of p53 (Pomerantz et
al., 1998; Zhang et al., 1998). The percentage of
polyploid cells is signicantly higher in p53 null and
pRb null MEFs compared to the p16Ink4a/p19ARF
double knockout MEFs (Di Leonardo et al., 1997a;
Khan and Wahl, 1998) which suggests that loss of
p16Ink4a and p19ARF is not functionally equivalent
to loss of either p53 or pRb.
A majority of normal primary human or mouse
broblasts treated with nocodazole arrest in the rst
G1 with 2N DNA content, suggesting that the signal
for arrest is generated prior to entry into mitosis (Di
Leonardo et al., 1997a; Khan and Wahl, 1998). The
signal for arrest generated by microtubule disruption,
therefore, appears to be independent of either aberrant
mitotic spindle assembly or chromosome segregation.
This raises the intriguing question of the nature of the
signal generated during G1 by microtubule disruption
that activates p53. The signal may be generated by
aberrant centrosome duplication, disruption of organelle and protein tracking along the microtubules,
changes in the equilibrium of monomeric/dimeric
tubulin to polymerized tubulin, or disruption of the
cell polarity in interphase cells. It remains to be
determined if p53 directly senses microtubule disarray
during G1 or if there are proteins upstream of p53 that
sense the disorganization and relay the signal for arrest
to p53. It is evident that the tumor suppressors p53
and pRb serve as a key link between cell cycle
progression and the integrity of the microtubule
network. Hence, loss of the ability to sense changes
in the cytoskeleton may be an additional factor that
contributes to the production of aneuploid variants
which are so frequently detected in cells undergoing
tumor progression.
Linking p53 activation with its subcellular localization
Although p53 gene mutation occurs in a substantial
fraction of human cancers, many tumors contain p53
that is functionally inactivated through other mechanisms. One such mechanism involves aberrant subcellular localization. Constitutive cytoplasmic localization
of wildtype p53 has been reported in inammatory
breast carcinoma (Moll et al., 1992), colorectal
adenocarninoma (Bosari et al., 1995; Sun et al.,
1992), undierentiated neuroblastoma (Moll et al.,
1995), hepatocellular carcinoma (Ueda et al., 1995),
and retinoblastoma (Schlamp et al., 1997), and is
associated with tumor metastasis and poor long-term
patient survival (Stenmark-Askmalm et al., 1994; Sun
et al., 1992). In addition, some viruses induce
uncontrolled cell proliferation through a strategy
involving p53 cytoplasmic sequestration. For example,
the adenovirus E1B-55-kDa protein anchors p53 in
cytoplasmic structures, reducing p53 transcriptional
activity (Blair Zajdel and Blair, 1988; Konig et al.,
1999; Sarnow et al., 1982; Yew and Berk, 1992;
Zantema et al., 1985). The hepatitis B virus HBx

protein localizes p53 to the cytoplasm and inhibits both

its transcriptional activity and its ability to mediate
apoptosis (Elmore et al., 1997; Feitelson et al., 1993;
Ueda et al., 1995; Wang et al., 1994, 1995).
Hepatocellular carcinoma is among the most common
malignancies world wide, with over 80% of patients
positive for Hepatitis B virus infection. As p53
mutations are found in only one-quarter to one-third
of these tumors (Ueda et al., 1995 and references
therein), cytoplasmic sequestration is clearly an
important mechanism of inactivating p53.
p53 cytoplasmic sequestration could result from its
anchorage to a cytoplasmic tether or by an imbalance
in nuclear-cytoplasmic shuttling. p53 has been shown
to bind multiple cytoplasmic proteins (such as,
ribosomes (Fontoura et al., 1997), hsc70 (Gannon
and Lane, 1991), hsp84 (Sepehrnia et al., 1996) and
various cytoskeletal elements (e.g. F-actin (Metcalfe et
al., 1999) and vimentin (Klotzsche et al., 1998)) which
may hold it in the cytoplasm and/or prevent its import
into the nucleus. However, the biological relevance of
the formation of these complexes remains to be
determined. Alternatively, it was shown recently that
in neuroblastoma cells, the rapid, energy-dependent
shuttling of p53 between the nucleus and cytoplasm is
altered so that hyperactive nuclear export results in net
cytoplasmic accumulation (Middeler et al., 1997; Moll
et al., 1995; Stommel et al., 1999). This result implies
that either an upstream signaling event which results in
nuclear export is constitutively active in these cells, or
that signals required for nuclear retention are missing.
Regardless of the mechanism, these data demonstrate
that the appearance of `cytoplasmically-sequestered'
p53 in these cells reects the inadequacy of static
microscopic observation of xed cells for the analysis
of a dynamic process. It remains to be determined if
the `cytoplasmically-sequestered' p53 in other cancers is
also a result of uncontrolled, hyperactive nuclear
export, or whether bona de cytoplasmic anchoring
mechanisms exist.
In normal, unstressed cells, p53 subcellular localization is variable and depends on the phase of the cell
cycle. p53 is predominantly nuclear from approximately mid G1 to the G1/S transition, then becomes
increasingly cytoplasmic as the cell progresses through
the cell cycle (David-Pfeuty et al., 1996; Shaulsky et al.,
1990a; Takahashi and Suzuki, 1994). This is consistent
with the observation that p53 is predominantly
cytoplasmic in mouse ES cells, which alternate
between S and M phases (Aladjem et al., 1998), In
response to various cytotoxic and genotoxic stresses,
post-translational modications increase p53 stability
and lead to nuclear accumulation (Fritsche et al., 1993;
Kastan et al., 1991). p53 nuclear accumulation is cell
cycle-dependent for some stresses, such as g-irradiation
(Komarova et al., 1997), while other stresses, such as
hydrogen peroxide and heat shock, induce p53 nuclear
translocation and accumulation independently of cell
cycle phase (Sugano et al., 1995). The observation that
the normal cell cycle controls of p53 subcellular
localization are overridden by some activators implies
the existence of stress-activated mechanisms for
regulating p53 nuclear accumulation. Analysis of girradiated cell fusions shows that p53 nuclear
accumulation is determined by a property of the
nucleus, indicating that this stress may produce

Regulation of p53 by diverse stresses

GS Jimenez et al

modications that inhibit p53 nuclear export rather

than accelerate its nuclear import (Komarova et al.,
1997). Therefore, tight regulation of p53 nuclear export
may be crucial for determining the kinetics, duration,
and magnitude of a stress response.
As p53 exerts many of its eects by transcriptional
regulation, its translocation from cytoplasmic sites of
synthesis to nuclear target genes is critical for the
elicitation of biological responses. Consequently, the
failure of p53 to be retained in the nucleus should
result in a defective p53 stress response. For example,
neuroblastoma and mouse embryonic stem cells have
cytoplasmic wildtype p53 and an impaired G1 arrest in
response to genotoxic stresses (Aladjem et al., 1998;
Moll et al., 1996). A temperature sensitive mutant p53
cannot suppress growth of transformed cells grown at
the 37.58C, the temperature at which it is predominantly cytoplasmic, but does so eectively at 32.58C
when it is nuclear (Martinez et al., 1991; Michalovitz et
al., 1990). In addition, a constitutively cytoplasmic
mutant derived from a Burkitt's lymphoma B-cell line
cannot suppress the transformation of primary rodent
broblasts by HPV16-E7 and H-ras, yet is fully
capable of transactivating p53-responsive genes and
inducing apoptosis when it is forced into the nucleus
through transient overexpression (Crook et al., 1998).
These results demonstrate the importance of nuclear
localization for p53 activity.
Some stresses, such as ribonucleotide depletion or
microtubule disruption, induce a reversible p53mediated cell cycle arrest (Khan and Wahl, 1998;
Linke et al., 1996). To enable renewed progression
through the cell cycle, the high levels of nuclear p53
which accumulate in a stress response must be reduced.
The proteasome-mediated degradation of p53 probably
occurs in the cytoplasm, since the export-inhibiting
drug leptomycin B stabilizes p53 (Freedman and
Levine, 1998). Therefore, the nuclear export of p53
prior to its degradation in the cytoplasm may be an
important component of the mechanism that regulates
p53 stability. Because an MDM2 mutant defective for
nuclear export is incapable of mediating the degradation of p53 (Freedman and Levine, 1998; Roth et al.,
1998), p53 degradation must occur via a mechanism
which depends on the export of MDM2. However, the
mere co-localization of MDM2 and p53 in the
cytoplasm may be insucient to induce p53 degradation, as cytoplasmic wildtype p53 in neuroblastoma
cells is immune to degradation by MDM2 (Moll et al.,
1996), and an MDM2 mutant defective for nuclear
import cannot facilitate the degradation of p53 (Tao
and Levine, 1999). It is tempting to speculate that
either or both proteins must acquire a modication in
the nucleus that allows p53 degradation. This is
consistent with the observation that constitutively
cytoplasmic p53 in neuroblastoma cells is not only
immune to proteasome-mediated degradation by
MDM2, but also by HPV E6 (Isaacs et al., 1999).
Compartmentalizing the steps that regulate p53
degradation could ensure that newly-synthesized p53
is not degraded prior to reaching the nucleus, and that
the half-life of activated, nuclear p53 is prolonged
when needed in a stress response.
Our understanding of p53 transcriptional activation
and stability has grown through years of analysis, but
investigation of the factors that regulate p53 sub-

cellular localization is embryonic by comparison. As

p53 is too large to passively diuse across the nuclear
pore, its subcellular distribution must be mediated by
the cell's protein import and export machinery.
Facilitated nuclear import of p53 occurs via three
lysine-rich nuclear localization signals (NLSs) in the Cterminus at amino acids 305322 (NLS I), 369-375
(NLS II), and 379384 (NLS III) (Liang et al., 1998;
Shaulsky et al., 1990b). NLS I appears to be the
primary NLS as mutation of this sequence alone
abrogates p53 nuclear import. In addition, the
mutation of the other two NLSs enhance the
cytoplasmic sequestration engendered by the NLS I
mutation, but by themselves have little eect on p53
localization (Shaulsky et al., 1990b). Little is known
about how p53 nuclear import may be regulated. A
protein of unknown function, spot-1, interacts with
NLS I, but the signicance of this binding remains to
be determined (Elkind et al., 1995). The cdc2/cdk2
phosphorylation site at S315 and the PCAF acetylation
site at K320 both lie within NLS I, and it is not
dicult to imagine that these modications could aect
association with a nuclear import receptor, especially
since the phosphorylation of NLSs has been reported
to regulate the import of other proteins such as SWI5
(Jans et al., 1995; Moll et al., 1991), Lamin B2
(Hennekes et al., 1993), v-Jun (Tagawa et al., 1995),
and CaM kinase II (Heist et al., 1998). However, since
an S315A mutation seems to have no eect on p53
nuclear localization (Liang et al., 1998), it remains to
be determined what role these modications may play.
The study of p53 nuclear export has become an
interesting topic of late, in part because of recent leaps
in our understanding of the mechanisms of nuclear
export, but also because of the discoveries of nuclear
export signals (NESs) in both p53 and MDM2
(Gorlich, 1998; Roth et al., 1998; Stommel et al.,
1999). p53 has a leucine-rich, rev-like NES in the Cterminus, which was shown to be functional through
multiple `gold standard' assays for export activity
(Stommel et al., 1999). For example, the p53 NES
can mediate the movement of p53 between nuclei in a
heterokaryon, it can facilitate the nuclear export of a
non-shuttling heterologous protein, and its activity is
sensitive to the export-inhibiting drug, leptomycin B
(Stommel et al., 1999). MDM2 has a similar rev-like
NES that mediates its nuclear export (Roth et al.,
1998). Because MDM2 binding to p53 is necessary for
p53 degradation, it was proposed that MDM2
functions as an NES-containing chaperone that
mediates p53 export to cytoplasmic proteasomes
(Freedman and Levine, 1998; Roth et al., 1998).
However, there is currently no direct evidence that
MDM2 is required for p53 nuclear export. On the
contrary, p53 has been shown to be fully capable of
nuclear export independently of MDM2 (Stommel et
al., 1999). On the other hand, it is clear that MDM2
nuclear import and export are required for p53
degradation (Freedman and Levine, 1998; Roth et al.,
1998; Tao and Levine, 1999). This is consistent with
the idea proposed above that MDM2 must receive a
modication in the nucleus that makes it competent for
p53 degradation. However, the role of MDM2 in p53
subcellular localization is still unclear.
A potentially important mechanism by which both
p53 subcellular localization and activation can be

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GS Jimenez et al

7662

coordinately regulated involves tetramerization. The

p53 NES lies within, and shares key residues with, the
tetramerization domain, such that the mutation of the
conserved leucines of the NES results in the abrogation
of both p53 export and tetramerization (Stommel et al.,
1999). Examination of three-dimensional structures of
the tetramerization domain reveals that the NES is
buried at the interface where two dimers combine to
make the active tetramer, and that it should be solventaccessible only in the monomeric or dimeric forms
(Clore et al., 1995; Jerey et al., 1995; Lee et al., 1994;
Mateu and Fersht, 1998; Waterman et al., 1995).
Consequently, a nuclear export receptor may be
incapable of binding tetrameric p53 due to NES
occlusion. Consistent with this hypothesis, a Cterminal peptide consisting of the p53 tetramerization
domain inhibits the hyperactive nuclear export observed in neuroblastoma cells (Ostermeyer et al., 1996;
Stommel et al., 1999). This appears to be due to the
binding of the peptides to the p53 NES such that when
these p53/peptide heterocomplexes enter the nucleus,
they may be incapable of nuclear export because the
binding surface recognized by the export receptor is
hidden by the peptide. Tetrameric p53 is most eective
at binding and transactivating p53 response elements,
and mutations which prevent tetramerization compromise DNA binding, diminish transactivation, and lead
to tumorigenesis (Friedman et al., 1993; Hainaut et al.,
1994; Halazonetis and Kandil, 1993; Hupp and Lane,
1994; Ishioka et al., 1995; Lomax et al., 1998; McLure
and Lee, 1998; Pietenpol et al., 1994; Tarunina et al.,
1996; Varley et al., 1996). Therefore, stress-induced
modications which allow p53 tetramerization to occur
may concurrently contribute to both the formation of
DNA-binding tetramers and to their retention in the
nucleus.
The modications which may mediate simultaneous
tetramer formation and NES inhibition are unknown,
but are likely to involve regulatory sites in the Cterminus. The C-terminus contains an inhibitory basic
domain that may be involved in intramolecular
interactions that prevent DNA binding, as peptide
binding to this region, or its deletion, enhances this
ability (Bayle et al., 1995; Hupp et al., 1995). Because
some C-terminal modications are induced by genotoxic agents (including K320 and K382 acetylation (Liu
et al., 1999; Sakaguchi et al., 1998), S376 dephosphorylation (Waterman et al., 1998), and S392
phosphorylation (Kapoor and Lozano, 1998; Lu et
al., 1998)), it is conceivable that they serve to neutralize
the inhibitory basic domain, thus enabling p53 to bind
DNA. These modications may induce structural
alterations which allow tetramer formation (Sakaguchi et al., 1997) and may consequently aect nuclearcytoplasmic shuttling. In addition, S376, S378, and
S392 are phosphorylation sites for PKC, PKA, and
CK2 respectively. Each of these kinases has been
implicated in regulating nuclear-cytoplasmic shuttling
of other proteins, such as diacylglycerol kinase
(Topham et al., 1998), dorsal (Briggs et al., 1998;
Drier et al., 1999), and SV40 T-antigen (Hubner et al.,
1997). In fact, PKC inhibitors induce both p53 DNA
binding and nuclear accumulation (Chernov et al.,
1998), both of which correlate with tetramer formation.
A number of the p53 C-terminal modications
mentioned above have been examined quite exten-

sively, yet much of the published data on their

relevance to p53 function is conicting. This is in
part due to the use of 32P in the assays, which could
potentially damage DNA and induce modications
which activate p53 and complicate interpretation of the
experiments. In addition, the EMSA or transactivation
assays typically used to assess p53 activity may be
inappropriate for determining the function of these
modications, as mutations at sites involved in
subcellular localization may be only indirectly relevant
to DNA binding. A more careful analysis of the role of
these and other sites in p53 nuclear-cytoplasmic
shuttling and tetramer formation may reveal how
temporal and spatial regulation of p53 activity can be
eectively coordinated in response to physiologically
and medically relevant stresses.
Additional unresolved problems
p53 activates both irreversible and reversible cell cycle
arrests, but the precise mechanisms leading to the
appropriate response remain to be elucidated. It is
possible that any stress that produces irreparable DNA
damage induces cell cycle exit, while conditions such as
ribonucleotide depletion or microtubule perturbation
produce a reversible arrest. It is reasonable to propose
that p53 activates dierent sets of downstream genes to
achieve the appropriate response, but the precise
spectrum of dierentially expressed genes is not yet
fully known. A more immediate challenge is to identify
whether dierent stresses activate p53 by producing
dierent modications in its primary structure.
The most intensively studied stress response involving p53 is that initiated by ionizing radiation. Initially,
an attractive model was that the signal transduction
system involved the Ku proteins and their catalytic
partner DNA-PK which phosphorylates the relevant
regions of the p53 N-terminus (Lees-Miller et al., 1992;
Shieh et al., 1997; Woo et al., 1998), but we now know
that this model is incorrect (Jimenez et al., 1999).
Rather, it seems more likely that the ATM and ATR
kinases are responsible for N-terminal p53 phosphorylation following ionizing radiation. However, these
proteins have no known Ku-like binding partners, and
so it remains to be determined how they `sense' the
presence of the relevant lesions. Furthermore, the
relevance of S15 phosphorylation has now been
brought into question by new evidence showing that
S20 plays a more important role in controlling p53
stability (Shieh et al., 1999; Unger et al., 1999).
Whether ATM and ATR act in a signal amplication
pathway to ensure that p53 is activated when as little
as only one lesion is present is also a matter for future
investigation.
The mechanism by which oncogenes activate p53 is
of substantial biological importance, as this stress may
provide the most profound selection for loss of p53
function during cancer progression. Furthermore, as
activated oncogenes can induce DNA breakage and
consequent genomic instability (Denko et al., 1994;
Felsher and Bishop, 1999; Fukasawa and Vande
Woude, 1997), loss of p53 function in the context of
an activated oncogene could signicantly accelerate the
rate of mutation accumulation and tumor progression.
It has been suggested that oncogenes activate p53 by a

Regulation of p53 by diverse stresses

GS Jimenez et al

mechanism that does not involve DNA damage but

does involve activation of p19ARF via hyperproliferative
signals (Sherr, 1998). In light of the data indicating
that oncogenes can induce DNA damage, it is prudent
to revisit this proposal. If no damage is involved, it will
be important to ascertain whether stresses which do
not induce DNA damage generally work though
p19ARF to activate p53.
Activation of p53 via p19ARF requires interaction of
p19ARF with MDM2 (Sherr, 1998). While p53 has been
the focus of studies to analyse stress induced covalent
modications, it is clear that these other binding
partners will need to be studied with equal scrutiny
for the dynamics of the activating mechanisms to be
fully understood. Finally, the mechanisms that control
the subcellular, or even subnuclear, localization of each
of these proteins are only starting to be studied.
Indeed, there are already competing hypotheses on the
role and relative importance of the nuclear export
signals in p53 and MDM2. While structural modifications in latent p53 are presumed to be necessary for
tetramer formation and consequent occlusion of the

intrinsic p53 NES, these remain to be identied, as do

the modifying enzymes. How the p53 tetramer is
destabilized to enable p53 nuclear exit and reversal of
a stress response, and whether MDM2 collaborates in
reversing p53 nuclear accumulation, are hotly debated
issues. As is true in any rapidly moving eld, the data
generated today only serve to pose more questions to
be answered tomorrow. Fortunately, the emergence of
many powerful analytical reagents such as sequence
and conformation specic antibodies, and strategies
such as expression arrays and high resolution
microscopes, should continue to provide answers at a
fast pace for the foreseeable future.

Acknowledgements
This work is supported by a postdoctoral fellowship from
the NIH (to GS Jimenez), a National Science Foundation
Graduate Fellowship (to JM Stommel), and grants from
the NIH and the G Harold and Leila Y Mathers
Charitable Foundation.

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