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Old 05-07-2008, 12:43 PM   #1
julierene
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Join Date: Dec 2005
Location: Illinois
Posts: 327
p53 regulation - Can anyone explain this?

Review
Mouse bites dogma: how mouse models are changing
our views of how P53 is regulated in vivo

GM Wahl*
,1
1


Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 N.
Torrey Pines Road, La Jolla, CA, USA
* Corresponding author: GM Wahl, Salk Institute for Biological Studies, Gene
Expression Laboratory, 10010 N. Torrey Pines Road, La Jolla, CA 92037,
USA. Tel: 858-453-4100x1255; Fax: 858-457-2762; E-mail: wahl@salk.edu
Received 04.1.06; accepted 20.2.06; published online 31.3.06
Edited by G Melino
Abstract
P53 is a transcription factor that can cause cells to be
eliminated by apoptosis or senescent-like arrest upon its
activation by irreparable genetic damage, excessively
expressed oncogenes, or a broad spectrum of other stresses.
As P53 executes life and death decisions, its activity must be
stringently regulated, which implies that it is not likely to be
controlled by a simple regulatory mechanism involving a
binary on–off switch. This brief review will summarize a
subset of the new information presented at the 10th P53
workshop in Dunedin, New Zealand in November 2004 as well
as very recent publications that provide new insights into the
molecular regulators of P53. Data emerging from mouse
models provide a fundamentally different view of how P53 is
regulated than suggested by more traditional in vitro
approaches. The differences between cell culture and mouse
models demonstrate the importance of preserving stoichiometric
relationships between P53 and its various regulators to
obtain an accurate view of the relevant molecular mechanisms
that control P53 activity.
Cell Death and Differentiation


(2006) 13, 973–983.
doi:10.1038/sj.cdd.4401911; published online 31 March 2006
Keywords:


p53 activation; mouse models; TP53
Abbreviations:


TAD1, transactivation domain
Introduction
Twenty-five years ago, a 53–54 kDa cellular protein now
referred to as P53 was reported to interact with viral
oncoproteins.1–5 Initial studies suggested that P53 functioned
as an oncogenic protein. Interestingly, recent studies that
modeled two such mutations in the mouse suggested that
they ‘gained function’ by binding to and attenuating the
functions of the P53-related proteins P63 and P73.6 This
provides one mechanism by which some P53 mutants
function as oncogenes. However, structural gene mutations
that disable P53 function, or that could engender dominantnegative
properties, have been reported in about 50% of
human cancers, including those that contribute most to
morbidity and mortality (e.g., see http://www-p53.iarc.fr/
index.html). Clear loss of both p53 alleles has been reported
in a significant fraction of human cancers.7 Together, these
data demonstrate that p53 is an important tumor suppressor.
8,9


Importantly, a substantial fraction of human tumors
express wild-type p53, but its function is compromised by
aberrant expression of proteins that negatively regulate it,
such as Hdm2 and HdmX (Hdm2 or HdmX designate the
human proteins, and Mdm2 and MdmX the mouse; the
corresponding genes are hdm2, hdmx, mdm2, mdmx).10–13
(Below, for simplicity, these regulators are collectively
referred to as Mdm2 or MdmX.) Together, the data show that
the P53 pathway is disabled in the majority, if not all, human
cancers.
P53 is an unstable transcription factor that can regulate
numerous downstream targets to induce permanent or
reversible cell cycle arrest, apoptosis, and DNA repair.14,15
While P53 has also been reported to induce apoptosis by
nontranscriptional mechanisms,16–18 substantial evidence
indicates that its transcriptional functions are critical for
effective tumor suppression.19
Signals elicited by stresses including DNA damage (e.g.,
see, see, Huang et al.,20 Kastan et al.,21 and Wahl and
Carr22), short or abnormally structured telomeres,23,24 highlevel
oncogene signaling,25–28 hypoxia,29 and glucose availability
30


activate P53. Cells that encounter such conditions
either arrest cell division or die if they possess a functional P53
pathway. By contrast, if P53 is dysfunctional, cells proliferate
when exposed to oncogenic stimuli, unrepaired DNA damage,
or metabolic perturbations that induce chromosome instability.
31,32


Thus, by eliminating P53 function, key controls to
prevent cell cycle entry under inappropriate, potentially
genome destabilizing conditions are lifted, allowing outgrowth
of the genetically unstable variants that fuel tumor progression.
Since P53 output has the potential to kill cells, stringent
regulatory mechanisms have evolved to prevent its errant
activation, as well as to allow for rapid activation when
appropriate. However, the regulatory mechanisms that enable
the P53 pathway to generate different transcriptional responses
to different stimuli in different tissues remain to be
defined. There is significant medical importance to having a
clear idea of P53 regulatory mechanisms as 3 million human
cancers per year are estimated to contain a wild-type
P53 protein whose function is attenuated. Understanding
the mechanisms underlying such negative regulation
affords a substantial opportunity for therapeutic intervention.
This possibility has been realized with the development
of agents such as cis-imidazolines (e.g., Nutlin 3A) that
interfere with Mdm2–P53 interactions to activate P53 in cells
with overexpressed Mdm2.33 Below, I review data from cell
Cell Death and Differentiation (2006) 13,


973–983
&


2006 Nature Publishing Group All rights reserved 1350-9047/06 $30.00
www.nature.com/cdd
culture and mouse models that provide a current view of
mechanisms that keep P53 off in unstressed cells,
allow its activation in response to conditions that produce
double-strand breaks, and then turn off P53 upon stress
abatement.
Integration of P53 Structure with
Regulation
P53 structure suggests many potential levels at which its
transcription function could be regulated (Figure 1). Transfection
studies suggest that P53 contains one N-terminal
transactivation domain (TAD1) comprising the first 40 amino
acids,34 and a second (TAD2) consisting of amino acids
43–63 that is revealed upon inactivation of TAD1.35 A potential
conformational switch may be contained within a domain
comprising proline-X-X-proline motifs between the TAD and
the large central DNA-binding domain.36–39 A C-terminal
oligomerization domain is necessary to form tetramers.40
These tetramers not only constitute the most active transcriptional
form of P5340–47 but also conceal the dominant nuclear
export signal that lies within the oligomerization domain.48 The
lysine-rich C-terminus constitutes a domain implicated in both
positive regulation by post-translational modifications involving
acetylation,49–52 methylation,53,54 and sumoylation,55,56
and negative regulation by ubiquitylation


57 or neddylation.58
The C-terminus has also been proposed to function as an
allosteric regulator.59–61 However, recent data make it more
likely that this domain enables P53 to bind nonspecifically to
DNA to increase the rate at which specific P53 response
elements are detected through linear diffusion.62,63
The N-Terminal TAD
Post-translational P53 modifications have been proposed to
induce structural alterations to stabilize the protein and to
effect the chromatin modifications needed for transcriptional
activation. The extreme N-terminal TAD has residues critical
for the binding of both the coactivators needed for transactivation,
and the E3 ubiquitin ligase MDM2 that contributes most
significantly to determining the abundance and short half-life
of P53 in unstressed cells.19 Indeed, mutation of just two
amino acids within this domain (L22Q, W23S in human, and
L25Q, W26S in mouse; referred to as P53QS below)
generates a stable but largely inactive P53 as the mutant
can neither interact with Mdm2 (and presumably MdmX,
which has a similar P53-binding domain) nor with the required
coactivators.
One attractive model is that DNA damage activates the
ATM kinase to induce a phosphorylation cascade beginning
with human ser15 (i.e., h-ser15), equivalent to mouse ser18
(m-ser18), which triggers the subsequent phosphorylation of
h-thr18 (m-thr21) culminating in phosphorylation of h-ser20
(m-ser23).64 These residues constitute part of a helical
domain that interacts with a hydrophobic pocket in the
Mdm2 N-terminus.65 In vitro studies with peptides from this
region indicate that thr18 phosphorylation can significantly
destabilize this structure, leading to Mdm2 dissociation.66
Transfection studies suggest that h-ser20 phosphorylation
significantly destabilizes the Mdm2/P53 interaction,67 while
others indicate that h-ser15 phosphorylation increases the
affinity for p300/CBP.68,69 These events together have been
suggested to stimulate P53 transactivation function. By
contrast, other studies showed that P53 mutants in which all
serine residues in the entire protein were changed to alanine
Figure 1


A competition model for positive and negative regulation. This is a very simplified view of p53, the N-terminal binding region for Mdm2 and MdmX, as well as
for p300/CBP. The structure of the p53 alpha-helical region binding within the hydrophobic cleft of Mdm2 is shown. The N-terminal region of MdmX that binds p53 is very
similar. The positions of S15, T18, and S20 are shown as they have been implicated in altering the N-terminal structure when phosphorylated to either decrease Mdm2
(MdmX?) binding or increase association with p300/CBP. Also shown in simplified form is the introduction of either positive or negative post-translational modifications to
multiple C-terminal lysines by either the HATs or by the E3 Mdm2. The N-terminal residues L22W23 that have been shown to prevent Mdm2 binding and severely reduce
transactivation when mutated to Q22 and S23 are shown in the primary structure and in their locations deduced from crystallographic analyses. See the text for more
details and references
P53 activation


in vitro and in mouse models
GM Wahl
974
Cell Death and Differentiation
displayed wild-type stability and transactivation.


19 Of great
significance, while mouse mutants generated by homologous
recombination to encode m-ser18ala appeared to exhibited
tissue-specific defects in p53 target gene activation, its
capacity to suppress spontaneous tumor formation remained
intact.70–72 Mice expressing the ser23ala mutation also
exhibited modest, tissue-specific deficiencies, but not the
substantial destabilization and functional inactivation that
would have been predicted were phosphorylation of this
residue critical for reducing Mdm2 binding.73,74 These data
are most consistent with N-terminal phosphorylations contributing
to modulating tissue-specific responses rather than
serving as an on–off switch as occurs in Drosophila.75,76
The interpretation of the significance of N-terminal phosphorylation
on P53 function also depends on whether P53
contains two TADs. If it does, then mutations in m-ser18 and
m-thr21 might only inactivate the first one, but preserve the
function of the second to allow some level of transactivation.
The question of whether P53 contains more than one
functional TAD was raised at the Dunedin meeting. The
human L22QW23S mutant was found by transfection analysis
to retain some residual function, and residues 43–63 were
subsequently identified as a second potential TAD (see
above). Recent data indicate that the second TAD mediates
activation of the proapoptotic IGFBP3 gene, and that the basic
C-terminus inhibits this function.77 This interpretation is based
on transfection studies, and it is important to emphasize that
the function of the second TAD is revealed only in the context
of P53 mutants deleted for TAD1, which contains the Mdm2
binding site. Thus, TAD1 mutants should encode a very stable
P53 variant capable of binding chromatin constitutively (see
below). The idea that the C-terminus is inhibitory to P53
function is inconsistent with other studies indicating that it
mediates linear diffusion to augment the ability of P53 to find
its response elements (see below). Therefore, it remains to be
determined whether the second putative TAD can contribute
to P53 functions in the context of a structurally wild-type P53
with compromised function for TAD1 while preserving stability
control by Mdm2.
A second approach to define the importance of transactivation
for P53-mediated tumor suppression, and to assess the
relative contribution of each P53 TAD in vivo, uses homologous
recombination to generate mice with inactivating
mutations in TAD1. Mice expressing a p53 with the mouse
equivalent of the L22QW23S mutations (i.e., L25QW26S,
referred to as p53QS) that largely inactivate human TAD1
have been generated by three groups.78–80 Two previous
studies showed that the p53QS mutant appeared to be
completely devoid of function.78,79 However, we now know
that the targeting construct we used to generate this mutant
contained an additional alanine to valine mutation at codon
135 in the DNA-binding domain (Ala135 to Val135). The
Val135 mutation also generates a temperature-sensitive
allele.81 Unfortunately, a second study did not attempt to
produce mice expressing p53QS, but did generate differentiated
ES cells and reconstituted thymus expressing this
allele.78 The data from both our studies and those of Chao
et al


.,78 as well as our more recent comparisons of the p53QS
alleles with Ala135 (p53QSA) or Val135 (p53QSV),


81 show
that the p53QS mutations themselves generate p53 molecules
devoid of detectable transcriptional activity, ability to
induce cell cycle arrest or apoptosis, or suppress tumor
formation under the conditions tested. In stark contrast to this
conclusion, data presented at the Dunedin meeting showed
that an independently generated p53QS with Ala135 is
embryonic lethal in heterozygotes.80 As no homozygous
p53QSA


embryos were obtained, the source of the lethality
remains to be determined. It is possible that the P53QSA
protein could stabilize and activate the remaining wild-type
P53, that the mutant allele could retain transcription function in
the proposed second TAD, or that another mechanism such
as that described below could underlie the observed lethality.
Our recent studies provide another way of envisioning
lethality generated by a stable, but transcriptionally inert
transcriptional regulator.81 We carefully compared the properties
of endogenous p53QSV with exogenously expressed
p53QSA (introduced into p53-null MEFs by lentiviral infection,
and clones expressing the same levels of P53QSV and
P53QSA proteins were identified and analyzed). We detected
no transactivation activity of either mutant on genes such as
p21


, mdm2, and Puma, but, consistent with the results from
the Attardi lab, we did detect bax transactivation by our
p53QSA


. However, bax activation by either our p53QSV or
QSA MEFs or those from the Attardi lab was only twice that
observed in p53-null cells.Wehave yet to observe induction of
cell cycle arrest, apoptosis, or suppression of xenograft
formation by p53QSA or p53QSV.81 p53QSA and p53QSV
are, as expected, extremely stable proteins (T1/248 h). There
is, however, one very important difference between P53QSA
and P53QSV proteins. While p53QSA binds p21, mdm2,
Puma


, and Noxa promoters constitutively and at levels equal
to that of activated wild-type p53, p53QSV binds these
promoters far less efficiently. Interestingly, we found that the
p53QSA generated in the Attardi lab is at least five times as
abundant in MEFs as p53QSV in MEFs derived from the mice
we generated, even though both proteins are equivalently
stable and p53 mRNA synthesis is similar for each allele.82
Based on all the available data, we propose the following
explanations for the embryonic lethality exhibited by the mice
produced by Johnson et al.80 First, the very high level of the
p53QSA protein in their mice could exhibit low activity in
particular cell types under specific conditions in vivo due to its
constitutive chromatin binding and weak transactivation
mediated by the second TAD. Another interpretation suggested
by our recent data suggests a transactivationindependent
mechanism of lethality. That is, although
p53QSA lacks measurable transactivation function, we
speculate that its accumulation to high levels, combined with
its tight and constitutive binding to chromatin, could interfere
with cellular transcription, replication, or other DNA-associated
molecular events to engender lethality.
The C-Terminus and p53 Regulation
The data summarized above lead us to suggest that there is
one dominant TAD in the N-terminus, and that, as deduced
from in vitro studies, there are residues within it that are
essential for the control of both P53 stability and transactivation.
A model that is accepted by most of the field has emerged
P53 activation


in vitro and in mouse models
GM Wahl
975
Cell Death and Differentiation
in which the binding of coactivators competes for binding of
the negative regulators Mdm2 and MdmX to the N-terminus.
This sets up a competition for competing modifications such
as acetylation and ubiquitylation of key highly conserved
lysines (six in human, seven in mouse) in the extreme
C-terminus.51
As described above, the C-terminus is likely to contribute to
P53 function by increasing the efficiency with which it finds its
specific response elements. Indeed, elegant studies presented
at Dunedin by C Prives showed that the C-terminus
enables p53 to engage circular DNA molecules, regardless of
whether they have a p53 response element. Deletion of the
C-terminus reduces association with random DNA, as found
earlier in studies of chromatin association.83 Once P53
engages random DNA, the rate at which it detects specific
P53 response elements is increased by linear diffusion.63
However, neither acetylation nor phosphorylation of the
C-terminus affected linear diffusion or target gene transactivation.
63,83
These


in vitro observations raise the question of whether Cterminal
modifications affect P53 function in vivo. Human P53
mutants in which all six highly conserved C-terminal lysine
residues were mutated to arginine to prevent post-translational
modifications including ubiquitylation and acetylation
proved to be stable and more active than wild-type P53.84,85
However, it is difficult to recreate the normal stoichiometric
relationships between P53, Mdm2, and MdmX using cotransfection
of P53 with Mdm2 into human cancer cell lines that
contain other alterations that could affect p53 function.
I presented data at the Dunedin meeting obtained from mice
generated by homologous recombination to encode p53 with
arginine substituted for the seven C-terminal lysines that are
analogous to the corresponding residues in human p53 (i.e.,
p537KR;86 also see87 for a study that generated a p53 allele in
which six lys were changed to arg but mice were not
produced). We developed this model to determine the impact
of C-terminal modifications on P53 regulation. While the in
vitro data suggested this mutation should be lethal if
homozygous, we observed normal Mendelian transmission
and no evidence of early mortality, sex bias, or developmental
abnormalities.86 Surprisingly, mouse P537KR and wild-type
P53 exhibit the same half-life, and like wild-type P53, p537KR
is degraded by Mdm2-mediated proteasome-dependent
proteolysis. In vitro analyses showed that P537KR can be
ubiquitylated by Mdm2 after transfection, but the pattern was
not the same as that of wild-type P53. As other studies
indicate that lysines other than in the C-terminus may control
P53 stability,88 it is formally possible that alternative lysine(s)
are targeted for ubiquitylation when those in the C-terminus
are not available. However, as P537KR has precisely the same
half-life as wild-type P53, it seems unlikely that Mdm2 could
engage widely separated lysines with equal efficiency.
Furthermore, while Mdm2 can polyubiquitylate itself, it can
only polyubiquitylate P53 at multiple lysines. This means that
if P53 needs to be polyubiquitylated for degradation, this must
require the action of an E4 ubiquitin ligase, such as p300.89 It
is difficult to reconcile the identical half-lives of these two
proteins with the observation that Mdm2 is sufficient to
meditate its own polyubiquitylation, while P53 requires both
Mdm2 and an E4. Thus, our data raise the intriguing possibility
that P53 degradation may merely require association with
Mdm2, and it is the self-ubiquitylation of Mdm2 that drives the
degradation of both proteins. However, this model seems
simplistic as Mdm2 can also bind to the P53-related proteins
P63 and P73, yet it does not mediate their degradation.90,91
Consequently, there must be other critical steps, such as
correct presentation of the substrate to the proteasome, or
engagement of linking molecules such as hHR23 to effect
substrate degradation.92,93 Clearly, more research is needed
to resolve the precise mechanism by which Mdm2 induces
P53 degradation, and to reveal how Mdm2 selectively
degrades p53.
We also investigated the activity of P537KR in cell culture
and in thymocytes in vivo. P537KR and wild-type P53 proved to
be experimentally indistinguishable in terms of their activation
kinetics, ability to induce target genes, cell cycle arrest, and
apoptosis in MEFs. However, P537KR was activated at lower
doses of ionizing radiation in mice when freshly explanted
thymocytes were analyzed. Importantly, this apparently
increased activity of P537KR was also observed in vitro when
MEFs were grown according to a 3T3 passage protocol. We
observed identical initial growth rates of MEFs expressing wild
type and P537KR, but MEFs encoding P537KR entered a
senescent state from which immortal variants did not arise.
This contrasts with MEFs encoding wild-type P53 that entered
crisis, but emerged as immortal variants. These data suggest
that while the conserved C-terminal lysines and associated
modifications are not essential for P53 control, they are likely
to fine-tune P53 activity, and to ensure that the magnitude of a
stress responses is appropriate. This fine-tuning is likely to be
critical for optimal lifespan of metazoans to guarantee that
cells that undergo numerous divisions, or at risk of stress
exposure, do not experience errant P53 activation.
The Proline-Rich Domain (PRD) and Mdm2
Interaction
P53 is exquisitely sensitive to Mdm2 expression levels. For
example, mice expressing a hypomorphic mdm2 allele
produced 30–50% of the normal Mdm2 protein level.94 This
resulted in precocious P53 activation in lymphoid tissue and in
some epithelial cells. Recent studies showed that mdm2þ/heterozygous mice were far more resistant to the development
of lymphoid tumors induced by expression of the Eu-Myc
transgene.


95 Finally, earlier onset and increased cancer
predisposition has been reported in premenopausal women
in whom a single-nucleotide polymorphism changes a T to aG
in the mdm2 promoter.96 This base change creates a binding
site for the ubiquitous transcription factor Sp1 adjacent to a
response element for the estrogen receptor, which apparently
together contribute to two- to four-fold elevated Mdm2 levels
that attenuate P53 function. Together, these data clearly show
that Mdm2 abundance is an important determinant of the
output of the P53 stress response pathway.
One way to modulate the impact of Mdm2 on P53 function is
to regulate the efficiency with which it binds P53. As stated
above, N-terminal phosphorylation may participate in this, but
its importance appears more significant in specific cell types.
Another region that may affect Mdm2 binding consists of a
P53 activation


in vitro and in mouse models
GM Wahl
976
Cell Death and Differentiation
loosely conserved PRD between the TAD and the DNAbinding
domain. This region contains a variable number of
PXXP motifs, where P¼proline and X¼any amino acid.
PXXP motifs provide potential interaction sites with proteins
containing src homology 3 motifs, and the PXXP domains of
P53 have been reported to interact with proteins including
Mdm2,38,39,97 p300,98 WWOX1,99 and the corepressor
mSin3a.100 Early studies showed that deletion of the PRD in
human P53 alter P53 transactivation in such a way as to
preserve the apoptotic function, while compromising cell cycle
arrest.101 Initial studies suggested that PRD deletion reduced
the affinity of P53 for apoptotic target genes,102,103 but
analyses performed at lower, more natural expression levels,
revealed reduced regulation of a broader spectrum of target
genes.104
Recent analyses have provided a potential molecular
mechanism for the compromised functionality of P53 lacking
the PRD. DNA damage can induce phosphorylation of
multiple threonine residues in threonine–proline motifs. This
creates sites that can be bound by the prolyl isomerase Pin1,
which can then induce cistrans isomerization of the adjacent
proline. Prolyl isomerization has been reported to reduce
Mdm2 binding, and to stabilize and activate P53.36,37,39
Deleting the prolines, or mutating the threonine to nonphosphorylatable
alanine, prevents the required prolyl isomerization,
and P53 damage responses appear to be compromised
in Pin1-deficient MEFs. A recent twist on this story was added
by Ygal Haupt at the Dunedin meeting, when he reported that
phosphorylation of thr81 is required to recruit Pin1, and that
Pin1 recruitment is required for efficient association with the
damage-activated kinase Chk2.39 Chk2 recruitment induces
phosphorylation of ser20, which then reduces affinity for
Mdm2, allowing P53 activation. Importantly, some humans
with the Li–Fraumeni cancer predisposition syndrome have
been reported to have chk2 mutations,105,106 while others
encode mutant p53 with proline 82 replaced by leucine (i.e.,
pro82leu).107 The latter data suggest the importance of pro82
as an important contributor to P53 function in vivo. Consistent
with this interpretation, Haupt described transfection studies
showing that P53 Pro82Leu accumulated less efficiently after
ionizing radiation, and exhibited reduced ability to activate a
p21 promoter-driven luciferase reporter.39 These data suggest
that the PRD may be important for regulating P53 output
by fine-tuning Mdm2 binding. One note of caution: earlier
studies by Dumaz and Meek showed that the impact of
deleting the PRD on P53 activation was critically dependent
on the ratio of Mdm2 to P53.97 Therefore, firm assessment of
the contribution of the PRD to P53 control will await the
generation and analysis of mice expressing p53 with a PRD
deletion, and others in which the prolines and threonines have
been mutated to rigorously evaluate the validity of the
conformational hypothesis advanced above.
New Thoughts on an Old Dogma
Over 36 000 papers addressing p53 function have been
published since its discovery 25 years ago. By contrast, a
fraction of this number has dealt with Mdm2 and MdmX, which
partly reflects their more recent discoveries.19,57,108,109
However, Mdm2 and MdmX are clearly critical negative
regulators of P53, and their functions are nonoverlapping
since deletion of either engenders early embryonic lethality.
110–114


This raises the possibility that important aspects of
P53 control remain to be elucidated due to the paucity of
attention focused on Mdm2 and MdmX and proteins that
control their functionality, such as Arf,115,116 gankyrin,117,118
and the ubiquitin-specific protease HAUSP (herpes virusassociated
ubiquitin-specific protease) (Li et al.,119,120 and
Meulmeester et al.121; and see below). Another puzzle is why
this system requires the two similar RING domain proteins
Mdm2 and MdmX to each serve as a P53-negative regulator.
Below, I present a model I proposed at the Dunedin meeting in
which I divided p53 regulation into four phases, and accounts
for the kinetic relationships between p53, Mdm2, and MdmX
we had observed at that time. I will review recent unpublished
and published data relevant to this model (Figure 2).
Phase 1: Homeostasis in unstressed cells
P53 is maintained at low levels and in an inactive state in
unstressed cells. This can be achieved in several ways. First,
Mdm2 can bind to P53 to mediate proteasomal degradation.
122,123


Second, Mdm2 can bind to the P53 TAD to prevent
recruitment of coactivators that access the same site.124
Third, as described by Moshe Oren


125 in Dunedin, Mdm2 can
monoubiquitinate the histones located in the vicinity of the P53
response element when a P53–Mdm2 complex engages
chromatin, and Mdm2 can also monoubiquitinate histones in a
P53-independent manner when it is overexpressed. Less
appreciated is the contribution of MdmX to attenuating P53
function in unstressed cells. This could be substantial as
MdmX is produced constitutively and is very stable (see
Marine and Jochemsen108,109 for reviews). The molecular
abundance of Mdm2 relative to MdmX in nonstressed and
stressed cells remains to be determined. Thus, P53 is held at
bay in unstressed cells by interactions with the two negative
regulators, Mdm2 and MdmX.
What then controls the levels of Mdm2 and MdmX? The
Mdm2 level in unstressed cells is determined by a number of
factors including, but not limited to the following: (1) P53-
dependent transactivation of the Mdm2 gene (see Lahav
et al


.125 for recent kinetic modeling); (2) mitogen-dependent
activation of factors such as Erk that also transactivate
Mdm2;126 (3) mitogen-dependent post-translational
modifications that modulate Mdm2 stability (e.g., see Ashcroft
et al


.127 and Gottlieb et al.128); and (4) interaction with
HAUSP.119–121,129 The factors that regulate MdmX abundance
have not been widely studied, but one that appears increasingly
important involves interaction with HAUSP.121 HAUSP
was first identified to participate in P53 control by Gu and coworkers
as a P53-associated protein.120 Inactivating HAUSP
by siRNA or by homologous recombination in HCT116 cells
resulted in Mdm2 and MdmX destabilization.120,121,130 The
importance of HAUSP in regulating MdmX level is demonstrated
by the lack of detectable MdmX in HAUSP-deficient
cells.121 As eliminating HAUSP greatly destabilized both
Mdm2 and MdmX, P53 became stabilized and activated.130
These observations imply that HAUSP interaction with, and
deubiquitination of, Mdm2 and MdmX contributes significantly
P53 activation


in vitro and in mouse models
GM Wahl
977
Cell Death and Differentiation
to their steady-state levels in unstressed cells. As MdmX has
a longer half-life than either Mdm2 or P53, it is reasonable to
speculate that MdmX may be the preferred substrate for
HAUSP-mediated deubiquitination.
To summarize regulation of Phase 1: Mdm2 accumulates
as a consequence of transcriptional activation by P53 and
mitogen-activated factors, as well as by ‘stabilization’ through
HAUSP interaction. While Mdm2 can ubiquitinate MdmX,
MdmX is deubiquitinated and stabilized via interaction with
HAUSP. The aggregate levels of Mdm2 and MdmX determine
both P53 abundance and transcriptional activity. Mdm2-
mediated ubiquitination of histones may provide an additional
means of controlling P53 transcription functions.
Phase 2: Early activation
DNA damage leads to the activation of ATM and other kinases
within minutes, as well as phosphorylation of N-terminal
serines that may modulate association with Mdm2/X and
coactivators. However, of equal importance, these kinases
also rapidly phosphorylate Mdm2 and MdmX.131–134 We
showed that phosphorylation of MDM2 by damage kinases
mediates its accelerated degradation early in the damage
response.135 Studies from the Yuan and Chen labs showed
that DNA damage also leads to MdmX degradation,132,136 and
we now know this requires the RING and acidic domains and
ubiquitin ligase activity of Mdm2.131,137–139
A picture of what triggers the accelerated degradation of
Mdm2 early in the damage response, and what converts
MdmX from a stable to an unstable protein is now emerging.
Recent work from the Shiloh and Jochemsen labs show that
the damage-induced phosphorylations on Mdm2 and MdmX
destabilize both proteins by preventing them from interacting
with HAUSP.121,134 Another study provided a somewhat
different explanation that phosphorylation increased association
between Mdm2 and MdmX.140 Regardless of which
mechanism is correct, the data taken together imply that DNA
damage triggers accelerated degradation of both negative
regulators. However, early in the DNA damage response in
normal fibroblasts and epithelial cells, it appears that MdmX
levels do not decline immediately, and that P53 target genes
are activated weakly. Indeed, full activation takes between 1
and 2 h.135 The next section provides one explanation of this
delay.
Phase 3. Full activation
It is clear that full P53 activation occurs only after a lag of about
2h.135 Damage kinases are active over this time, P53 remains
phosphorylated at h-ser15, and presumably Mdm2 and Mdmx
are also phosphorylated over this interval. One of the first
genes activated by P53 is Mdm2, but early in the damage
response, the Mdm2 that is made is unstable, likely because it
is phosphorylated and cannot interact with HAUSP. Mdm2
Figure 2


Models for attenuating and destabilizing p53, and for activating and stabilizing it in response to stress. P53 regulation is presented in four phases as described
in detail in the text. It is important to note that the relative numbers of molecules of P53, Mdm2, MdmX, and HAUSP are not known. Neither is the extent to which Mdm2
and MdmX exist as homo- and heterodimers, and whether this changes as Mdm2 and MdmX levels increase or decline during a stress response. *Indicate damagekinase
phosphorylations that prevent interaction with HAUSP. See the text for a detailed explanation of the model and additional limitations
P53 activation


in vitro and in mouse models
GM Wahl
978
Cell Death and Differentiation
does accumulate over time, however, and this correlates with
progressively decreasing MdmX levels. This raises the
possibility that P53 activates Mdm2 so that its increased
abundance ensures MdmX degradation. This model predicts
that conditions that lead to P53 activation in the absence of
activation of damage kinases should also lead to MdmX
degradation due to the elevated Mdm2 abundance. As shown
by Lubo Vassilev at the Dunedin meeting, P53 can be fully
activated by Nutlin3a, which prevents Mdm2 from binding to
P53.141 Indeed, we found that Nutlin3a results in substantial
increases in Mdm2 abundance, and that MdmX levels decline
in parallel (J Stommel, M Wade, M Tang, and G Wahl,
unpublished data). Importantly, we also showed that proteasome
inhibitors prevent P53 activation after DNA damage,
even though P53 was phosphorylated in its N-terminus.135 By
contrast, Nutlin addition during proteasome inhibitor treatment
enabled P53 activation by reducing Mdm2 (and presumably)–
MdmX interaction with P53.135 Together, the data indicate
that P53 activation requires Mdm2 and Mdmx phosphorylation
to decrease HAUSP interaction with both proteins, the net
effect of which is to increase Mdm2-mediated self-ubiquitylation
and ubiquitylation of MdmX. This decreases the levels of
Mdm2 at early time points and as a consequence activates
P53, resulting in increased Mdm2 transactivation. The
increased levels of Hdm2 can then degrade MdmX, resulting
in full activation of P53. In this model, P53 activation requires a
positive feedback loop


in which increasing Mdm2 abundance
titrates the amount of MdmX degradation to assure the finetuning
of the timing and magnitude of the P53 transcriptional
response.
Phase 4: Attenuation
The components of this system also provide the potential to
attenuate P53 signaling should a stress dissipate or DNA
damage be repaired. I suggest this could occur in the following
way. ATM and P53 ser15 phosphorylation return to background
levels within 4–6 h of induction of DNA damage with a
low dose of the radiomimetic agent neocarzinostatin.135 In
normal human fibroblasts, Mdm2 levels are high at 4 h, and
start to decline thereafter, along with a parallel decrease in
P53 transactivation. In light of the data summarized above, it
is reasonable to propose that the damage-phosphorylated
Mdm2 pool is replaced with a nonphosphorylated pool as a
consequence of new synthesis and diminished abundance of
activated ATM. The elevated levels of Mdm2 should be able to
interact with and inactivate P53 by the mechanisms described
above. Furthermore, in the absence of activated damage
kinases, MdmX will be allowed to interact with HAUSP,
leading to deubiquitination and stabilization. This provides a
second barrier to continued P53 activation.
Perspectives
The integration of recent data described above provides a
new way of conceptualizing P53 regulation. Homeostasis is
maintained by the well-known negative feedback loop. A new
twist to the loop is provided by the idea that the activation of
Mdm2 during a stress response actually creates a positive
feedback loop in which Mdm2 mediates MdmX degradation to
achieve full P53 activation. This elegant system enables
gradual and tightly controlled increases in P53 activity rather
than an all-or-none response that might be created were the
system solely controlled solely by phosphorylation. Finally,
the reservoir of Mdm2 created by P53-mediated activation
enables attenuation of the P53 response since most of the
MdmX is eliminated in the activation phase.
This model accounts for most of the major regulators known
to operate in this system, and it is consistent with the kinetics
of the response in the normal fibroblasts and epithelial cells
we have analyzed. It is clear that the system is very sensitive
to small changes in the stoichiometric relationships between
HAUSP, Mdm2, MdmX, and P53, and it is likely that proteins
that regulate Mdm2 function, such as Arf or Gankyrin, may
enable finer tuning of the system, or be required for responses
to stressors other than DNA damage. Clearly, an important
goal for the near future will be to determine the precise
numbers of each of these molecules in normal and neoplastic
cells, and to determine how differences in stoichiometry affect
the dynamics and magnitude of stress-activated responses.
We have begun a cursory analysis of cell lines with wild-type
P53, and have found substantial variations in the relative
abundance of each molecule. This may well explain the
attenuation of the P53 response in the numerous cancers
expressing wild-type P53.
An important unresolved question concerns the molecular
mechanism that switches Mdm2 target specificity from P53 to
MdmX. Clearly, one component of the mechanism is
determined by whether HAUSP interacts with and deubiquitinates
Mdm2 and MdmX. However, as described above, the
binding of Mdm2 to P53 may be all that is needed for P53 to be
degraded. As N-terminal phosphorylation of P53 seems
unlikely to prevent Mdm2 from binding to P53, we need to
determine whether P53 stabilization involves preventing
Mdm2 from interacting with P53, or whether additional factors
are involved. As one example, perhaps proteins that connect
Mdm2 to the proteasome, such as hHR23, comprise part of
the switch.92,93
The excitement in this field has grown with the successful
isolation of a variety of compounds such as the Nutlins that
can activate wild-type P53 in some tumor lines. What we now
need to determine is whether the different expression patterns
of the key proteins that control P53 function will impact on the
utility of these agents, or if the molecular signatures of cancer
cells can be used to define subsets of patients most likely to
respond to P53 activator therapies. Of significance, as MdmX
has emerged to play an increasingly important role in P53
activation, the optimal drugs for p53 activation clearly need to
target both Mdm2 and MdmX to prevent both from interacting
with and inhibiting P53.


__________________
Jan04: Bilateral Mastectomy at age 28
Initial DX: Left Breast: IDC 2cm, Grade 3, HER2+3, 0 Nodes +, ER/PR-. Right Breast: Extensive DCIS ER-/PR+; Stage 1-2a
Feb04-Apr04: 4 AC, dose dense
Aug 04: 4 Taxotere
Dec 05: Bone and Liver METS; Stage 4. Carboplatin/Taxol/Herceptin. DX with Li-Fraumeni Syndrome
Apr 06: NED, maintenance Herceptin
Apr 07: CA1503=14; masses in liver; Xeloda/Tykerb
Nov 07: NED, Tykerb maintenance
Sept 08: Liver mets again, on Tykerb/Xeloda again, CA=19 and 27
Nov 08: Progression, Tykerb/Gemzar, CA=25
Dec 08: Progression, Herceptin/Navelbine, CA=40, 57, and 130
Jan 09: Progression in bone, recession in liver, Herceptin/Carbo/Abraxane CA=135
June 09: CA27/29=24, chemo break
Sept 09: Progression, CA=24, waiting on clinical trial (4 weeks no treatment)
Nov 09: now have brain mets, trial "on hold", getting 14 WBR treatments starting 11/2/09
Dec 09: possible start on p53 trial
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Old 05-07-2008, 06:16 PM   #2
hutchibk
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we need one of the propellerheads to translate for us!
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Brenda

NOV 2012 - 9 yr anniversary
JULY 2012 - 7 yr anniversary stage IV (of 50...)

Nov'03~ dX stage 2B
Dec'03~
Rt side mastectomy, Her2+, ER/PR+, 10 nodes out, one node positive
Jan'04~
Taxotere/Adria/Cytoxan x 6, NED, no Rads, Tamox. 1 year, Arimadex 3 mo., NED 14 mo.
Sept'05~
micro mets lungs/chest nodes/underarm node, Switched to Aromasin, T/C/H x 7, NED 6 months - Herceptin only
Aug'06~
micro mets chest nodes, & bone spot @ C3 neck, Added Taxol to Herceptin
Feb'07~ Genetic testing, BRCA 1&2 neg

Apr'07~
MRI - two 9mm brain mets & 5 punctates, new left chest met, & small increase of bone spot C3 neck, Stopped Aromasin
May'07~
Started Tykerb/Xeloda, no WBR for now
June'07~
MRI - stable brain mets, no new mets, 9mm spots less enhanced, CA15.3 down 45.5 to 9.3 in 10 wks, Ty/Xel working magic!
Aug'07~
MRI - brain mets shrunk half, NO NEW BRAIN METS!!, TMs stable @ 9.2
Oct'07~
PET/CT & MRI show NED
Apr'08~
scans still show NED in the head, small bone spot on right iliac crest (rear pelvic bone)
Sept'08~
MRI shows activity in brain mets, completed 5 fractions/5 consecutive days of IMRT to zap the pesky buggers
Oct'08~
dropped Xeloda, switched to tri-weekly Herceptin in combo with Tykerb, extend to tri-monthly Zometa infusion
Dec'08~
Brain MRI- 4 spots reduced to punctate size, large spot shrunk by 3mm, CT of torso clear/pelvis spot stable
June'09~
new 3-4mm left cerrebellar spot zapped with IMRT targeted rads
Sept'09~
new 6mm & 1 cm spots in pituitary/optic chiasm area. Rx= 25 days of 3D conformal fractionated targeted IMRT to the tumors.
Oct'09~
25 days of low dose 3D conformal fractionated targeted IMRT to the bone mets spot on rt. iliac crest that have been watching for 2 years. Added daily Aromasin back into treatment regimen.
Apr'10~ Brain MRI clear! But, see new small spot on adrenal gland. Change from Aromasin back to Tamoxifen.
June'10~ Tumor markers (CA15.3) dropped from 37 to 23 after one month on Tamoxifen. Continue to monitor adrenal gland spot. Remain on Tykerb/Herceptin/Tamoxifen.
Nov'10~ Radiate positive mediastinal node that was pressing on recurrent laryngeal nerve, causing paralyzed larynx and a funny voice.
Jan'11~ MRI shows possible activity or perhaps just scar tissue/necrotic increase on 3 previously treated brain spots and a pituitary spot. 5 days of IMRT on 4 spots.
Feb'11~ Enrolled in T-DM1 EAP in Denver, first treatment March 25, 2011.
Mar'11~ Finally started T-DM1 EAP in Denver at Rocky Mountain Cancer Center/Rose on Mar. 25... hallelujah.

"I would rather be anecdotally alive than statistically dead."
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