Mouse bites dogma: how mouse models are changing our views of how p53 is regulated in vivo

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. SIMILAR CONTENT BEING VIEWED BY

OTHERS TISSUE SPECIFICITY AND SPATIO-TEMPORAL DYNAMICS OF THE P53 TRANSCRIPTIONAL PROGRAM Article 08 February 2023 _TP53_: THE UNLUCKIEST OF GENES? Article Open access 23 October 2024

SHIFTING THE PARADIGMS FOR TUMOR SUPPRESSION: LESSONS FROM THE P53 FIELD Article 08 June 2021 INTRODUCTION Twenty-five years ago, a 53–54 kDa cellular protein now referred to as P53 was

reported to interact with viral oncoproteins.1, 2, 3, 4, 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 dominant-negative 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, 11, 12, 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, 17, 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 high-level oncogene signaling,25, 26, 27, 28 hypoxia,29 and glucose availability30 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 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, 37, 38, 39 A C-terminal

oligomerization domain is necessary to form tetramers.40 These tetramers not only constitute the most active transcriptional form of P5340, 41, 42, 43, 44, 45, 46, 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, 50, 51, 52 methylation,53, 54 and sumoylation,55, 56 and negative regulation by ubiquitylation57 or neddylation.58 The C-terminus has also been

proposed to function as an allosteric regulator.59, 60, 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 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, 71, 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, 79, 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. We have 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 (_T_1/2>8 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

transactivation-independent 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 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 C-terminal 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 _mdm2_+/− 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 a G 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 loosely conserved PRD between the TAD and the DNA-binding

domain. This region contains a variable number of PXXP motifs, where P=proline and X=any 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 _cis_–_trans_ 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,

111, 112, 113, 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 virus-associated 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 Oren125 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, 120, 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 co-workers 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 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, 132, 133, 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, 138, 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 2 h.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 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 fine-tuning 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. ABBREVIATIONS * TAD1:

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dispensable for transcriptional activation and apoptosis. _J. Biol. Chem._ 279: 53015–53022. CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS I wish to thank all the members

of my lab, past and present, for their hard work and many animated discussions that made it possible to develop the models shown. Although Jayne Stommel is no longer in the lab, her

demonstration of damage-mediated destabilization of Mdm2 as an important early event in P53 activation and stabilization caused me to look at the problem very differently. The work of

Crystal Lee and Franck Toledo, who have also left recently, pointed out the importance of MdmX as a critical negative regulator of P53 transactivation in an _in vivo_ setting. Finally,

current members Kurt Krummel, Mark Wade, Vivian Wang, Ee-Tsin Wong, Leo Li, Stephanie Kinkel, and Mengjia Tang either provided data or suggestions that improved or clarified the manuscript.

This work has been supported by grants from the NCI (CA61449, CA48405) to GMW, MT, KK, and MW. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Salk Institute for Biological Studies, Gene

Expression Laboratory, 10010 N. Torrey Pines Road, La Jolla, CA, USA G M Wahl Authors * G M Wahl View author publications You can also search for this author inPubMed Google Scholar

CORRESPONDING AUTHOR Correspondence to G M Wahl. ADDITIONAL INFORMATION Edited by G Melino RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Wahl, G. Mouse

bites dogma: how mouse models are changing our views of how P53 is regulated _in vivo_. _Cell Death Differ_ 13, 973–983 (2006). https://doi.org/10.1038/sj.cdd.4401911 Download citation *

Received: 04 January 2006 * Accepted: 20 February 2006 * Published: 31 March 2006 * Issue Date: 01 June 2006 * DOI: https://doi.org/10.1038/sj.cdd.4401911 SHARE THIS ARTICLE Anyone you share

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Nature SharedIt content-sharing initiative KEYWORDS * p53 activation * mouse models * TP53