p53 has been studied intensively as a major tumour suppressor that detects oncogenic events in cancer cells and eliminates them through senescence (a permanent non-proliferative state) or apoptosis. Consistent with this role, p53 activity is compromised in a high proportion of all cancer types, either through mutation of the TP53 gene (encoding p53) or changes in the status of p53 modulators. p53 has additional roles, which may overlap with its tumour-suppressive capacity, in processes including the DNA damage response, metabolism, aging, stem cell differentiation and fertility. Moreover, many mutant p53 proteins, termed ‘gain-of-function’ (GOF), acquire new activities that help drive cancer aggression. p53 is regulated mainly through protein turnover and operates within a negative-feedback loop with its transcriptional target, MDM2 (murine double minute 2), an E3 ubiquitin ligase which mediates the ubiquitylation and proteasomal degradation of p53. Induction of p53 is achieved largely through uncoupling the p53–MDM2 interaction, leading to elevated p53 levels. Various stress stimuli acting on p53 (such as hyperproliferation and DNA damage) use different, but overlapping, mechanisms to achieve this. Additionally, p53 activity is regulated through critical context-specific or fine-tuning events, mediated primarily through post-translational mechanisms, particularly multi-site phosphorylation and acetylation. In the present review, I broadly examine these events, highlighting their regulatory contributions, their ability to integrate signals from cellular events towards providing most appropriate response to stress conditions and their importance for tumour suppression. These are fascinating aspects of molecular oncology that hold the key to understanding the molecular pathology of cancer and the routes by which it may be tackled therapeutically.

INTRODUCTION: THE MANY ROLES OF THE p53 PROTEIN

A man for all seasons”–Robert Bolt

The p53 tumour-suppressor protein functions mainly, but not exclusively, as a tightly regulated transcription factor that encompasses both transactivation and transrepression activities [14]. p53 has the capacity to regulate the expression of several hundred genes, many of which are involved in mediating or regulating cell growth, division, survival and/or programmed cell death. Under normal homoeostatic conditions, p53 is a short-lived protein that is regulated mainly through changes in its protein stability [5]. However, in response to a range of stress stimuli, including activated oncogenes (hyperproliferation), ribosomal stress and various forms of DNA damage (Figure 1), p53 is induced, essentially by blocking its degradation, leading to increases in its cellular levels. Elevated p53 levels promote, in a context-dependent manner, biological outcomes of growth arrest, senescence or apoptosis, which are widely accepted tumour-suppression mechanisms that block uncontrolled proliferation of transformed cells or eliminate them completely. The ability of p53 to regulate the onset of apoptosis is also mediated partly at the mitochondrion through its transcription-independent function as a pro-apoptotic BH3 (Bcl-2 homology domain 3)-only-like factor [6]. p53 has also been demonstrated to down-regulate the generation of new blood vessels (angiogenesis) [7], an activity that is thought to make a key contribution to tumour suppression.

Stress stimuli that lead to p53 induction

Figure 1
Stress stimuli that lead to p53 induction

A wide range of cellular stress stimuli induce p53, leading to co-ordinated changes in gene expression and various biological outcomes, depending on the cell type and the type, intensity and duration of the activating stress. Those events that induce p53 through the DNA-damage-response pathways are highlighted on the left-hand side in lilac. Regulation of some biological events may occur in a homoeostatic manner mediated by basal or low levels of p53. ROS, reactive oxygen species.

Figure 1
Stress stimuli that lead to p53 induction

A wide range of cellular stress stimuli induce p53, leading to co-ordinated changes in gene expression and various biological outcomes, depending on the cell type and the type, intensity and duration of the activating stress. Those events that induce p53 through the DNA-damage-response pathways are highlighted on the left-hand side in lilac. Regulation of some biological events may occur in a homoeostatic manner mediated by basal or low levels of p53. ROS, reactive oxygen species.

The main focus of p53 for many years has been its role in cancer as a tumour suppressor, earning the title ‘Guardian of the genome’ [8]. However, it is now clear that p53 additionally regulates a range of genes essential for wide-ranging cellular processes, often when present at basal non-induced levels (Figure 1). For example, p53 is able to control implantation and, consequently, fertility by regulating expression of LIF (leukaemia-inhibitory factor) [9], an observation that explains the reduced litter sizes frequently seen in p53-null mice. p53 can regulate the proliferation and differentiation of stem cells [10,11]. It can also function to restrict longevity and promote the aging process [12,13]. It has also been established that p53 plays a key protective role in promoting skin pigmentation through its ability to stimulate expression of POMC (pro-opiomelanocortin), a multi-component precursor containing α-MSH (α-melanocyte-stimulating hormone) [14]. Stimulation of melanin-synthetic enzymes by α-MSH, which leads to activation of the key melanocyte transcription factor MITF (microphthalmia-associated transcription factor), constitutes a major line of defence against harmful UV rays in sunlight and, ultimately, against the development of skin cancer. There is also evidence that p53 can moderate innate immune responses through its antagonism of NF-κB (nuclear factor κB) signalling [15,16].

p53 is now also known to play a critical role in regulating metabolism. Through its ability to regulate genes involved in intermediary metabolism and mitochondrial respiration, p53 can reduce flux through the glycolytic and pentose phosphate pathways, and stimulate mitochondrial function [4,17,18]. p53 can thus favour mitochondrial oxidative phosphorylation as the principal means of ATP production and minimize the synthesis of substrates needed for growth and cell division. p53-dependent regulation of these genes also acts as a barrier to the Warburg effect [19], in which oncogenic processes promote aerobic glycolysis and flux through the pentose phosphate pathway. p53 additionally regulates the IGF-1 (insulin-like growth factor 1)/mTOR (mammalian target of rapamycin) pathways [17] and thus governs the routes by which proliferation, survival and energy metabolism are controlled.

In contrast with these protective effects, there is growing evidence that p53 activity can contribute to various pathologies [20]. For example, in animal models for Parkinson's disease, loss of DJ-1 expression leads to increased levels of p53 and consequently to neuronal cell death [21]. Similarly, impairment of the activity of parkin, a protein which acts, in part, as a transcription factor that can repress p53, is thought to exacerbate cell death in Parkinson's disease patients [22]. During the development of Huntington's disease, induction of p53 through the DNA-damage pathways may contribute to cell death [2326]. There is also evidence associating p53 induction with neuronal death in Alzheimer's disease (see [27] and references therein). In addition to neurodegenerative disease, p53-dependent apoptosis arising from ischaemia and myocardial infarction contributes to the tissue injury associated with these conditions [2831]. Also, the induction of p53 through the DNA-damage pathways is a major contributor to the side effects of radiotherapy and chemotherapy. Additionally, recent evidence, again from mouse models, indicates that p53 can mediate some of the pathological effects of chronic inflammation that are relevant to age-related muscle wasting, septic shock and Parkinson's disease [32]. Taken together, these lines of evidence indicate that inappropriate activation of p53 can have a detrimental effect on biological outcome.

p53 STRUCTURE/FUNCTION

Made up of various Parts, one perfect Harmony”–Nicholas Brady (Ode to St Cecilia)

From a structural perspective, p53 contains several domains that are essential for mediating its various functions (Figure 2). Two contiguous transactivation domains, termed TAD1 and TAD2 respectively, are located at the N-terminus. TAD2 overlaps with a proline-rich domain that makes important contributions to repression, apoptosis and the response to γ-irradiation [33]. The central region or ‘core’ domain encompasses the site-specific DNA-binding function of p53 and is essential for transactivation, the repression of some genes and tumour suppression. The C-terminal region contains sequences required for nuclear localization, non-specific DNA binding and regulation. The regulatory region undergoes a variety of PTMs (post-translational modifications) that play key roles in controlling p53 turnover and in fine-tuning its activity (see below) [3437]. The C-terminus also contains a tetramerization domain which mediates the formation of homo- and hetero-tetramers. This domain is crucial for transactivation and tumour suppression of wild-type p53, and for the interaction of various p53 isoforms (discussed below).

Modular structure of p53

Figure 2
Modular structure of p53

The p53 protein is shown schematically, highlighting important functional domains. Boxes I–V represent the highly conserved regions of p53 first highlighted by Soussi et al. [253]. TAD1 and TAD2 are the transcriptional activation domains, TET is the tetramerization domain, REG is the C-terminal regulatory region, NES is the nuclear export sequence, and NLS is the nuclear localization sequence.

Figure 2
Modular structure of p53

The p53 protein is shown schematically, highlighting important functional domains. Boxes I–V represent the highly conserved regions of p53 first highlighted by Soussi et al. [253]. TAD1 and TAD2 are the transcriptional activation domains, TET is the tetramerization domain, REG is the C-terminal regulatory region, NES is the nuclear export sequence, and NLS is the nuclear localization sequence.

p53 is a member of a family that includes p63 and p73, both of which are structurally very similar to p53 [38] and are each able to activate transcription of many p53 target genes. Biologically, p63 and p73 each have highly specific roles that are distinct from those of p53. For example, p63 plays a major role in the development of squamous epithelia, whereas p73 is indispensable for neuronal development. Moreover, all three family members co-operate in the regulation of maternal reproduction [9]. In mouse models, p63 and p73 can also contribute to tumour suppression through various mechanisms and act co-operatively with p53 [38,39].

Another feature that is shared by all three family members is that their respective genes each encode a series of products, or isoforms (including full-length proteins), generated through alternative promoters, internal translational start sites and alternative splicing mechanisms. Cross-talk between different isoforms of the various family members can occur through protein–protein interactions and can have a major impact on biological functions and specificities associated with these genes [40]. Accordingly there is now a shift in the perception that events involving p53 may not simply be the result of the action of a single protein but occur as the result of an interactive group of isoforms, the presence of which may occur selectively depending on the cell type and cellular or stress conditions [41].

p53 AND TUMOUR SUPPRESSION

The needs of the many outweigh the needs of the few…or the one”–Spock (The Wrath of Khan)

p53 provides a critical barrier to the development of cancer by blocking proliferation or eliminating cancer cells. p53-null mice are susceptible to spontaneous tumour formation, whereas the removal of p53 in mouse models for various specific cancers leads to rapid tumour development and death (e.g. see [4244]). Consistent with a tumour-suppressive role, mutation of the TP53 gene has proven to be the most common event in the development of human cancer and occurs at high frequency [45]. In those cancers that lack TP53 mutation, other alterations can occur (e.g. in the levels or activities of upstream regulators) that prevent the activation of p53 [46].

Strikingly, restoring wild-type p53 function in animal models can regress cancer development and significantly extend periods of survival [4749]. Establishing this principle has opened up a variety of approaches towards reactivating p53 as an anti-cancer therapeutic strategy [50]. Mouse models have also provided compelling evidence that p53-mediated tumour suppression can occur through apoptosis or senescence, and have revealed that the relevant importance of these tumour-suppressive mechanisms is likely to be tissue- or cell-type-dependent [43,48,49,5154]. Alternatively, in some instances, cell cycle arrest, senescence and apoptosis may be dispensable for tumour suppression, and other important cancer-relevant p53 activities such as metabolic regulation or antioxidant function may be required [5558].

Over the last two decades, a large number of p53-responsive genes have been identified that contribute towards mediating p53 downstream events [3,59]. [This list includes genes encoding the cyclin-dependent kinase inhibitor p21 (WAF1/CIP1) which mediates cell cycle arrest at G1/S-phase and G2/M-phase; GADD45 (growth-arrest and DNA-damage-inducible protein 45), 14-3-3-σ and Reprimo which down-regulate CDK1 (cyclin-dependent kinase 1) activation at G2/M-phase; CDC25 (cell division cycle 25), cyclin B1 and PLK1 (Polo-like kinase 1) which promote G2/M transition and are repressed by p53; BAX (Bcl-2-associated X protein), PUMA (p53 up-regulated modulator of apoptosis), NOXA and others that control the induction of apoptosis; and TIGAR (TP53-induced glycolysis and apoptosis regulator) which controls flux through the glycolytic pathway.] Curiously, however, p53-dependent transcription of a large number of its responsive genes is dispensable for the suppression of at least some cancers and only a small number of generally less well-characterized p53-responsive genes (but including BAX) are now thought to have a crucial involvement in this process [5558]. Notably, transcription of this subset of tumour-suppressive genes does not require the involvement of the p53 TAD1 subdomain which is a major target for activation by the DNA-damage signalling pathways (see below) and which is required for induction of the ‘classical’ p53-responsive genes (e.g. p21, PUMA and NOXA) involved in growth arrest and apoptosis.

‘GAIN-OF-FUNCTION’ MUTANT p53 PROTEINS IN CANCER

The Dark Side of the Force is the pathway to many abilities some consider to be… Unnatural.”–Supreme Chancellor Palpatine (Star Wars Episode II: Attack of the Clones).

Since the early 1990s, the role of p53 in cancer has been firmly considered to be that of a tumour suppressor. However, for about a decade after its discovery in 1979, p53 had been studied in its then perceived capacity as an oncogene. In the last decade, it has become clear that mutation of the TP53 gene during cancer development does not simply result in the ablation of wild-type p53 tumour-suppressor activity, but that mutations acquired by TP53 can give rise to proteins that do, indeed, encompass oncogenic functions [60,61].

The majority of TP53 mutations that arise during cancer development are missense mutations that lie within the part of the gene that encodes the site-specific DNA-binding domain, supporting the idea that DNA binding and transcription are critical for tumour suppression. Although there are six ‘hotspots’ that represent approximately 30% of all of the recorded mutations, missense changes in any of a wide range of other codons within this region are sufficient to ablate or significantly impair DNA binding. DNA-binding mutants come in essentially two forms: ‘contact’ mutants, where key residues that make highly specific contacts with p53REs (p53-responsive elements) in genes are substituted; and ‘conformational’ mutants that cause conformational shifts in the core and thus disfavour DNA binding. Notably, many, if not most, mutations in the core domain leave the tetramerization domain intact, thereby allowing the mutant p53 protein to assemble into tetramers that can include wild-type p53 subunits encoded by the non-mutated allele. Consequently, the presence of a mutant p53 subunit(s) in the tetramer has an established dominant-negative effect on p53 site-specific DNA binding and transactivation function, and may weaken p53 activity as a tumour develops.

Additionally, and importantly, many mutant p53 proteins acquire new oncogenic functions that are now known to contribute to the development of cancer [6064]. Indeed, analysis of certain hotspot mutations in the context of mouse models has established that mutant p53 can bring about aggressive behaviour including genetic instability and the development of metastasis [6567]. Functional analysis in cultured cells has established that many GOF mutants drive or contribute to cancer-associated activities including survival, invasion, migration, proliferation, genomic instability and drug resistance [60], all of which are established hallmarks of cancer [68]. How these proteins function biochemically, and whether different mutants show different behaviours, is only clear in part. Notably, although the DNA-binding domain is dysfunctional in many of these proteins, they are still able to interact with other cellular components through unaltered domains including the tetramerization domain and the TADs (summarized in [62]). Thus mutant p53 proteins can influence cellular functions mechanistically by associating with certain transcription factors to stimulate expression of growth-promoting genes, aggregating with other transcription factors to squelch their activities, and interfering with the function of other p53 family members such as suppressing the anti-metastasis activity of p63 (see below) [62].

REGULATION OF p53 BY MDM2

Who will guard the guardians themselves?”–Juvenal (Satire VI)

p53 is maintained at low levels through ubiquitylation and proteasomal degradation mediated mainly by the RING-finger type E3 ligase, MDM2 (murine double minute 2) [6972]. There are six lysine residues in the C-terminus of p53 that act as major targets for ubiquitylation (Lys370, Lys372, Lys373, Lys381, Lys382 and Lys386) [73], but, importantly, MDM2 is also able to direct the ubiquitylation of other lysine residues in the p53 protein in vivo [74,75]. High levels of MDM2 promote polyubiquitylation of p53, leading to its degradation. However, in the presence of lower levels of MDM2, mono-ubiquitylation of p53 occurs and facilitates different events such as p53 nuclear export [76]. MDM2 and p53 act within a negative-feedback loop in which p53 transactivates MDM2 expression through the stronger of two promoters in the MDM2 gene, thus maintaining or increasing the levels of its negative regulator as is appropriate [77]. The significantly increased levels of MDM2 achieved following the induction of p53 are pivotal in restoring p53 to homoeostatic levels following removal of the inducing signal (see below).

Mutant p53 proteins are generally very stable in cancer cells and can be detected easily by immunohistochemistry. Curiously, just as with wild-type p53, mutant p53 is maintained at low levels in normal cells [78]. Whereas mutant p53 proteins are unable to transcriptionally induce MDM2, and thus maintain the feedback loop, the levels of MDM2 provided through the action of the weaker p53-independent promoter in the MDM2 gene are thought to be sufficient to keep p53 levels low in the absence of stress stimuli. Stabilization of mutant p53 is thus considered to be a feature of the developing cancer that, crucially, amplifies and underpins GOF activity and can be achieved, at least in animal models, through loss of Mdm2 or p16 [78]. However, it is not yet clear whether this reflects the mechanism of p53 stabilization in human cancer. Alternatively, the low levels of DNA damage that are characteristic of developing cancer cells have been proposed to induce p53, but where the p53 is mutant and is therefore unable to stimulate MDM2 expression and engage the negative-feedback loop, there is a failure in restoring homoeostatic p53 levels [79]. These findings have striking implications for signalling to p53 and suggest that the normal mechanisms that elevate wild-type p53 levels (such as the DNA-damage pathways that are induced following the treatment of cancer patients with genotoxic drugs) may also act to accentuate GOF activity (see below).

THE INTERACTION BETWEEN p53 AND MDM2

Mungojerrie and Rumpleteazer had a wonderful way of working together. And some of the time you would say it was luck…”–T.S. Eliot (Old Possum's Book of Practical Cats)

MDM2 and p53 interact sequentially through several points of contact (Figure 3). A hydrophobic cleft in the N-terminus of MDM2 serves as a docking site for three key hydrophobic residues in the N-terminus of p53: Phe19, Trp23 and Leu26 [80]. Association of p53 and MDM2 through this high-affinity interaction is thought to produce a conformational shift that allows an essential low-affinity contact between the central acidic domain of MDM2 and the Box IV/V region of p53 to occur [8184]; this ‘ubiquitylation signal’ in the p53 core is indispensable for the subsequent interaction of p53 with the RING domain of MDM2, leading to p53 ubiquitylation by recruited E2 ligase.

Concerted mechanism of p53 ubiquitylation by MDM2

Figure 3
Concerted mechanism of p53 ubiquitylation by MDM2

The p53 and MDM2 proteins are shown in schematic modular format. Interaction of the N-terminal TAD1 of p53 with the N-terminal hydrophobic pocket in MDM2 leads to a conformational shift that permits the acidic domain of MDM2 to associate with the so-called ‘ubiquitylation signal’ in the Box IV/V region of p53. This brings the C-terminal domain (CTD) of p53 into contact with the RING domain of MDM2 together with the E2 ligase, leading to ubiquitylation of p53. PRO, proline-rich domain.

Figure 3
Concerted mechanism of p53 ubiquitylation by MDM2

The p53 and MDM2 proteins are shown in schematic modular format. Interaction of the N-terminal TAD1 of p53 with the N-terminal hydrophobic pocket in MDM2 leads to a conformational shift that permits the acidic domain of MDM2 to associate with the so-called ‘ubiquitylation signal’ in the Box IV/V region of p53. This brings the C-terminal domain (CTD) of p53 into contact with the RING domain of MDM2 together with the E2 ligase, leading to ubiquitylation of p53. PRO, proline-rich domain.

MDM4, also known as MDMX, is a defective E3 ligase which is structurally related to MDM2 and which acts, in part, as a suppressor of p53-mediated transcription [8588]. MDM2 is considered to be a weak E3 ligase, but its ability to modify p53 can be enhanced through contact with a number of proteins, including dimerization between MDM2 and MDM4 through their respective RING fingers [89]. MDM4 can thus act as an important stimulatory partner for MDM2 that favours polyubiquitylation of p53 [89,90]. Importantly, the turnover of MDM4 and MDM2 itself is mediated by MDM2 [9194] {although there is evidence that other factors can regulate MDM2 turnover such as PCAF [p300/CBP (cAMP-response-element-binding protein-binding protein)-associated factor] [95]}.

In terms of regulating p53 transcriptional activity, MDM2 and MDM4 can sterically interrupt association of transcriptional proteins with p53 that is anchored on chromatin and recruit histone deacetylase activity to p53 and neighbouring histone proteins [96]. MDM2 can also perturb the conformation of the p53 core domain, thereby inhibiting its site-specific DNA-binding function [97]. Furthermore, by promoting mono-ubiquitylation of p53, MDM2 can expose a nuclear export signal on p53, leading to its translocation to the cytoplasm [98,99]. There is also evidence that MDM2 can inhibit TP53 mRNA translation indirectly by promoting the degradation of the ribosomal protein L26, an activator of TP53 mRNA translation [100]. Collectively, these activities work in a co-ordinated and complementary fashion to down-regulate p53 levels.

MDM2 is the most extensively studied p53 ubiquitin ligase and is essentially ubiquitous in cells and tissues. It is critical for p53 regulation as demonstrated by the early in utero lethality of mice lacking MDM2 expression [71,72]. There are, however, at least 15 additional ubiquitin E3 ligases which are able to regulate p53, but in a context- and possibly cell-type-dependent manner [101]. Moreover, p53 operates within several other feedback loops with other proteins, including some of these E3 ligases, which have a corresponding influence on its levels and function [102]. Similarly, MDM2 itself has a growing list of different partner proteins and substrates, many of which are relevant to cancer [103]. These findings indicate that signals that uncouple the p53/MDM2 interaction are likely to have wider and possibly selective outcomes and influences on switching on p53.

THE COMPLEXITY OF p53 REGULATION: POST-TRANSLATIONAL MODIFICATIONS, ISOFORMS, PROTEIN–PROTEIN INTERACTIONS AND CROSS-TALKING PATHWAYS

Layers. Onions have layers. Ogres have layers… You get it? We both have layers.”–Shrek

Although the relationship with MDM2 plays a central role in governing p53 levels and function, p53 activity is regulated and fine-tuned in a much wider context of control through various mechanisms mediated by protein–protein interactions, PTMs, differential expression of p53 isoforms and responsiveness to cross-talking pathways.

p53 has an extensive number of interacting partner proteins, including those through which its function is mediated (e.g. transcription factors) and those that operate by controlling p53 levels and/or activity. A comprehensive list of these proteins is given elsewhere [34,104]. Two major examples of partner proteins that are central to governing p53 induction are the ARF (alternative reading frame) protein, which stimulates p53 levels in response to hyperproliferative signals, and a select group of ribosomal proteins that induce p53 following ribosomal or nucleolar stress. Both of these mechanisms play key roles in the activation of p53 tumour-suppressor function and are discussed below.

In addition to ubiquitylation, p53 is subject to a wide range of other PTMs, including multi-site phosphorylation, acetylation, methylation and SUMOylation (these have been described in detail elsewhere [5,3436,105108]. Moreover, a recent publication, in which modifications on p53 in a standard cell line were analysed by MS (mass spectrometry), identified as many as 150 different PTMs of the protein, suggesting that the scope for modification is profoundly deeper and more complex than had previously been appreciated [109]. These modifications play important roles in regulating p53 levels, activity, protein–protein associations, subcellular localization and responsiveness to cross-talk emanating from other signalling pathways. This extensive list of modifications, taken together with the knowledge that the TP53 gene is now thought to encode up to 12 different isoforms, some or all of which themselves may undergo PTM, suggests that there is an eye watering number of permutations that may exist for p53 and, consequently, for its functional status at any given time and in any given biological context. Owing to this profound level of complexity, the following analysis focuses mainly on full-length p53 and on those well-characterized PTMs that have been established to play a major role in regulating p53 function.

THE MECHANISM OF p53 INDUCTION AND ACTIVATION: ESSENTIAL SHARED EVENTS AND STIMULUS-SPECIFIC PROCESSES

There are only a few notes. Just variations on a theme.”–John Lennon

The main event in the induction of p53, regardless of the initiating stimulus, is the uncoupling of p53 from degradation mediated by MDM2 [5]. This is achieved by various mechanisms depending upon the nature of the inducing signal and is discussed in some detail below [5]. p53 is also restrained by other regulators which act in concert with MDM2 (including MDM4) and which may also be modulated during p53 induction (Figure 4). Additionally, p53, MDM2 and MDM4 are subject to deubiquitylation mediated by the ubiquitin protease HAUSP (herpesvirus-associated ubiquitin-specific protease; also known as USP7 or ubiquitin-specific protease 7) [110116]. Under normal, unstressed, conditions, HAUSP activity is targeted selectively to p53 by the adaptor protein DAXX with the outcome of minimizing MDM2 auto-ubiquitylation but promoting p53 ubiquitylation and turnover [114] (Figure 5). There is evidence that in response to at least some stresses, the action of HAUSP on p53 is relieved and redirected, thereby contributing to p53 stabilization and increased turnover of its inhibitors.

Location of key DNA-damage-induced phosphorylation sites within the modular structures of p53, MDM2 and MDM4

Figure 4
Location of key DNA-damage-induced phosphorylation sites within the modular structures of p53, MDM2 and MDM4

The p53, MDM2 and MDM4 proteins are shown schematically, highlighting important functional domains. Those sites of phosphorylation that are directly relevant to the DNA damage (strand break) response are shown (P; yellow ellipses), together with the protein kinases(s) known to phosphorylate them. The sites of acetylation (Ac; red circle) and ubiquitylation (Ub; green circle) are also indicated. Comprehensive lists of the PTMs in these proteins are available elsewhere [34,88,105,108,189,254,255]. p53BD, p53-binding domain; Zn, zinc finger domain; other abbreviations are defined in Figure 2, Table 1 and the text.

Figure 4
Location of key DNA-damage-induced phosphorylation sites within the modular structures of p53, MDM2 and MDM4

The p53, MDM2 and MDM4 proteins are shown schematically, highlighting important functional domains. Those sites of phosphorylation that are directly relevant to the DNA damage (strand break) response are shown (P; yellow ellipses), together with the protein kinases(s) known to phosphorylate them. The sites of acetylation (Ac; red circle) and ubiquitylation (Ub; green circle) are also indicated. Comprehensive lists of the PTMs in these proteins are available elsewhere [34,88,105,108,189,254,255]. p53BD, p53-binding domain; Zn, zinc finger domain; other abbreviations are defined in Figure 2, Table 1 and the text.

The idea that p53 remains intrinsically active but kept in a transcriptionally inactive form through the binding of MDM2 and MDM4 has been termed the ‘anti-repression’ model [96]. To a large extent, this model describes accurately the condition stated above that activation of p53 can be achieved simply by blocking the action of its inhibitors. However, p53 is also subject to many regulatory events, particularly those entailing various PTMs, which can fine-tune its activity and biological outcome. This suggests that, for a p53 response that is appropriate to the cellular status and the needs generated by the stress stimulus, the uncoupling of its association with MDM2 and MDM4 is simply not enough.

The best-characterized mechanisms of p53 induction/activation are those initiated by hyperproliferative signals arising from oncogene action, ribosomal stress and various forms of DNA damage. The molecular bases of these mechanisms are as follows.

Induction of p53 in response to activated oncogenes

Activated oncogenes (such as Ras, Myc, E2F-1 and β-catenin) use various mechanisms to stimulate the levels of the ARF protein (also known as p14ARF), an inhibitor of MDM2 that is encoded by the alternative reading frame in the CDKN2A gene and overlaps with the cell-cycle-regulatory INK4A (p16) locus. Mechanisms of ARF induction include stimulation of ARF transcription, inhibition of ARF degradation and segregation of ARF from its targeting ubiquitin ligase, ULF (reviewed in [5]). ARF interacts directly with the central acidic domain of MDM2 and inhibits its ubiquitin ligase function. Recent evidence indicates that the binding site in MDM2 for ARF undergoes intramolecular association with the RING domain and functions as a critical stimulator of ubiquitin transfer. Mechanistically, therefore, ARF binding disrupts this interaction [117]. Additionally, the ARF protein resides in the nucleolus where it is able to sequester MDM2 in a physically separate compartment from nucleoplasmic p53. ARF also functions as a p53 activator by stimulating MDM2–MDM4 contact and thus promoting MDM4 degradation [118]. ARF can furthermore stimulate the phosphorylation and inactivation of the RelA subunit of the NF-κB transcription factor, leading to down-regulation of the ability of NF-κB to oppose the p53-mediated apoptosis [119]. From a cancer perspective, mice lacking a functional Arf gene develop multiple types of tumours at a high frequency [120]. Consistent with the mouse models, inactivation of the INK4A/ARF locus by mechanisms such as deletion, mutation or epigenetic silencing, is frequently observed in a variety of human cancers [121].

Induction of p53 in response to ribosomal stress

Ribosomal stress (also known as nucleolar stress) arises when the highly co-ordinated process of ribosome synthesis and assembly is interrupted [5,122]. This can occur through DNA damage within ribosomal genes, or through impaired rDNA expression or rRNA processing, with the outcome that there is an excess of ribosomal proteins over and above the amount needed for assembling new ribosomes. Specific ribosomal proteins (namely L5, L11, L23, S3, S7, S14 and S27) are then able to interact directly with MDM2 within overlapping regions in its central acidic and/or zinc-finger regions, leading to the inhibition of MDM2 ubiquitin ligase function and, consequently, the accumulation of p53. The binding of these ribosomal proteins may involve steric hindrance of the p53–MDM2–RING domain interaction, or, as is the case with the ARF protein [117], interfere with intramolecular acidic region–RING finger association. Alternatively, the binding of these proteins may reduce the flexibility of MDM2, thereby preventing it from adopting appropriate conformational changes. An added function of L26 is that it can bind to the 5′ UTR of TP53 mRNA and stimulate p53 translation [100]. Since Myc can promote ribosomal protein translation [123], it may also elevate p53 levels indirectly through this mechanism. The induction of p53 through the ribosomal pathway may also have a central role in the development of at least some types of cancer. In this respect, mice harbouring a cysteine to phenylalanine mutation in the zinc-finger domain of Mdm2 (Mdm2C305F), the region responsible for L5 and L11 binding, fail to respond to inducers of ribosomal stress, and also show significantly accelerated Eμ-Myc-induced lymphomagenesis. This important study highlighted the potential of cross-talk between the ribosomal protein- and ARF-dependent mechanisms of induction in triggering tumour suppression [124]. In contrast with this outcome, Mdm2C305F had no effect on the development of prostate cancer in a mouse model, indicating that the nucleolar stress pathway in cancer prevention is likely to be context-specific [125].

Induction of p53 by MDM2-targeted drugs

Since p53 tumour-suppressor function may be inhibited in some cancers through the overexpression or amplification of MDM2, a number of small-molecule MDM2 inhibitors have been developed with the therapeutic goal of reactivating p53 in such cancers [126]. The prototypic member of this group of compounds is Nutlin-3a (hereinafter Nutlin) which mimics the binding of p53 to the N-terminus of MDM2 and thus competitively blocks association with p53 [127,128]. The treatment of cells with Nutlin induces high levels of p53 protein and gives rise to a robust induction of p53-downstream gene expression and biological outcomes [5]. This level of activation can exceed and/or differ from the p53 response produced by other stimuli. For example, the NF-κB pathway, which, like p53, is activated by DNA damage, adopts several mechanisms to down-regulate p53 transcriptional activity, including sequestration of limiting transcription factors such as the histone acetyltransferases, CBP [KAT3A (lysine acetyltransferase 3A)] and p300 (KAT3B), that are crucial for mediating p53-dependent transactivation [129,130]. Given that compounds such as Nutlin act simply as pharmacological inhibitors of MDM2, they may not stimulate other regulatory events within the p53 pathway, or in parallel pathways, that happen in response to a natural stimulus (see, e.g., [131]). This may have a significant effect on the outcome of inducing p53 with such compounds. Accordingly, combining Nutlin treatment with other targeted molecular therapies, or conventional chemo- and radio-therapies, has been demonstrated to strengthen its effect on p53-dependent apoptosis (see, e.g., [132]).

Induction of p53 in response to DNA damage

The induction and activation of p53 through the DNA-damage pathways is orchestrated by two highly related protein kinases, ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia- and Rad3-related) which share overlapping substrate specificities and are activated in response to double- and single-strand breaks respectively. Whereas these two protein kinases initiate a series of events that activate and co-ordinate appropriate DNA-repair mechanisms, a key element of their function is the activation and integration of various signalling pathways leading to changes in the post-translational status of p53 itself (discussed below) and several of its direct or indirect regulators [5,34,35,88,105108] (Figure 4). As with other pathways that induce p53, the major focus of ATM and ATR is the inhibition of MDM2-mediated degradation of p53. What is additionally clear about this response is that, following the uncoupling of p53 from the actions of its inhibitors, a sequential and stimulus-dependent series of post-translational events in p53 occur that promote the recruitment, by p53, of key transcription factors, leading to chromatin remodelling and transcriptional activation.

The key event in activating p53 in response to DNA damage is the phosphorylation of MDM2, mediated principally by ATM [133,134]. The major site for phosphorylation by ATM is Ser395 in human MDM2 (Ser394 in mouse Mdm2), which is located towards the C-terminus between the zinc finger and RING finger domains [135] (Figure 4). Other residues in this region (Ser386, Ser407, Ser425, Ser429 and Thr419) can also be modified by ATM with similar functional consequences in cultured cells [136]. ATR can also phosphorylate Ser407 [137] and perhaps other residues in this region, given its shared substrate specificity with ATM. Biochemically, modification of one or more of these sites allosterically inhibits the formation of MDM2 dimers and/or higher-order structures that are mediated through the RING domain, with the outcome of blocking poly-, but not mono-, ubiquitylation of p53. Phosphorylation of MDM2 at these sites thus leads to stabilization of p53 and, consequently, deactivation of the negative-feedback loop. Additionally, these modifications disrupt the interaction between the acidic domain of MDM2 and the Box IV/V region of p53 that constitutes the ‘ubiquitylation signal’. ATM-dependent phosphorylation suppresses a further activity of MDM2, that of inducing an inhibitory conformational shift in the p53 DNA-binding domain [138]. Phosphorylation of MDM2 by the c-Abl protein kinase, which itself is activated by ATM, occurs at Tyr394 within this cluster and contributes to inhibition of MDM2 [139].

The key evidence supporting the dominance of this model in vivo was provided from the analysis of Mdm2S394A/S394A and Mdm2S394D/S394D knockin mice [133,134]. Mdm2S394A/S394A mice are born at Mendelian ratios and show basal levels of p53 and Mdm2 that are indistinguishable from those of wild-type mice. However, they are exceptionally radio-resistant and are thus unable to induce p53-dependent apoptosis in response to IR (ionizing radiation). IR-dependent p53 induction and downstream gene expression in the Mdm2S394A/S394A mice are weak compared with wild-type mice. However, the responses of cells to Nutlin are unaffected. Mdm2S394D/S394D mice, on the other hand, are phenotypically similar to wild-type mice, but show delayed attenuation of the p53 response to IR, consistent with the idea that dephosphorylation of this residue is required to restore homoeostasis.

Setting the bar for p53 induction

Other mechanisms (e.g. cross-talking pathways) can have an impact on the ability of these activation pathways to bring about a robust p53 induction. For example, survival signalling and/or oncogenic signalling via activated Akt leads to phosphorylation and activation of MDM2 with the outcome of increasing the threshold needed for a significant p53 response [140,141]. Likewise, elevated glucocorticoid levels (arising, for example, through psychological stress) phosphorylate and activate MDM2 via SGK1 (serum- and glucocorticoid-activated protein kinase 1) and may thus increase cancer susceptibility [142]. The NF-κB pathway can modify and/or compete for limiting cellular proteins such as p300 and CBP that p53 normally requires for its own downstream effects [129,130]. Activation of NF-κB in parallel to p53 induction, through a common stimulus such as DNA damage, can therefore have an impact on the p53 response. Furthermore, changes in levels of MDM2 expression in cells, for example, in individuals with the single nucleotide polymorphism SNP309, can also significantly affect p53 responsiveness and is associated with increased risk for certain cancers [143].

Regulation of p53 biochemical function in response to DNA damage

Crucially, although ATM/ATR-mediated MDM2 phosphorylation is key to uncoupling p53 from MDM2-mediated degradation, DNA damage stimuli initiate a broader series of events that underpin both the induction process, through co-ordinate inhibition of p53 negative regulators, and, importantly, activate p53 biochemical functions in a manner that is appropriate and proportional to the type, strength and duration of the stimulus. These events involve co-ordinated and temporal regulation, mainly through PTM, of p53, MDM2, MDM4 and other critical regulators.

The acidic domain of MDM2 contains a cluster of phosphorylated residues that are modified under homoeostatic conditions and contribute towards the turnover of p53 in the absence of stress stimuli [144146] (Figure 4). Curiously, loss of these phosphorylation sites has no effect on the ubiquitylation function of MDM2, but significantly impairs its ability to target p53 for degradation. (These findings established that different activities encompassed within the MDM2 protein can be regulated differentially.) Biochemical analyses suggest that these sites are modified in a sequential manner, through the combined action of the protein kinases CK1 and GSK3β (glycogen synthase kinase 3β) [147149]. In parallel to ATM-mediated events towards the C-terminus, IR leads to hypophosphorylation of this domain in a manner that is thought to contribute to uncoupling p53 from MDM2-mediated degradation [144]. The protein kinases that phosphorylate this region are abundant and normally active under unstressed conditions, consistent with a role in maintaining p53 degradation. Following a DNA damage signal, however, a mechanism is thought to occur in which activated DNA-PK (double-stranded DNA-activated protein kinase) mediates the phosphorylation and activation of Akt2 [150,151] which, in turn, leads to inhibition of GSK3β, decreased phosphorylation of the MDM2 acidic domain, and reduced p53 turnover. It is unclear whether this mechanism co-operates with the ATM-mediated interruption of the interaction between the MDM2 acidic domain and the Box IV/V region of p53 (described above).

MDM4 is also down-regulated in response to DNA damage, through phosphorylation of Ser342 and Ser367 by CHK2 (checkpoint kinase 2) and direct phosphorylation of Ser403 by ATM [91,94] (Figure 4). (In the UV response, phosphorylation of Ser367 is mediated by CHK1 [152].) Phosphorylation of Ser367 permits the binding of 14-3-3-γ which then leads to the ubiquitylation and degradation of MDM4 and inhibits its ability to co-operate with MDM2 in promoting p53 turnover [152155]. MDM4 phosphorylation also promotes inhibition of its interaction with the deubiquitinase HAUSP, leading to increased MDM4 ubiquitylation and degradation [113,155]. Mice encoding alanine substitutions at residues 342, 367 and 403 are radio-resistant and, although they show no increase in spontaneous tumour formation, they do exhibit increased sensitivity to Myc-induced tumorigenesis [156]. MEFs (mouse embryonic fibroblasts) and thymocytes isolated from these mice show impaired p53 induction following γ-irradiation or neocarzinostatin (NCS) treatment, supporting an in vivo role in regulating p53 levels. Like MDM2, MDM4 also undergoes DNA-damage-dependent phosphorylation by c-Abl in response to DNA damage. The two target residues, Tyr55 and Tyr99, are located in the p53-binding domain. Phosphorylation of one of these, Tyr99, impairs p53 binding and may thus contribute to p53 activation [157]. Dephosphorylation of an isoform of HAUSP (USP7S) may also underpin these events in p53 induction [158]. In response to DNA damage, ATM-dependent activation of PPM1G (Mg2+/Mn2+-dependent protein phosphatase 1G) leads to dephosphorylation of Ser18, a CK2-targeted site, and destabilization of USP7S, thereby contributing to MDM2 turnover and p53 activation (Figures 4 and 5).

Mechanism of p53 activation in response to DNA damage

Figure 5
Mechanism of p53 activation in response to DNA damage

p53 is shown together with its main regulators as a complex before activation (left) and following DNA damage-induced phosphorylation and dissociation (right). The mechanism is described in detail in the text.

Figure 5
Mechanism of p53 activation in response to DNA damage

p53 is shown together with its main regulators as a complex before activation (left) and following DNA damage-induced phosphorylation and dissociation (right). The mechanism is described in detail in the text.

Sequential PTMs in p53 regulate association with MDM2 and interaction with transcription factors

Within the p53 protein itself, a number of pivotal PTMs occur that not only underpin the regulation of the p53–MDM2 interaction discussed above, but also contribute significantly to the fine-tuning of p53 transcription function. Key among these is the phosphorylation of p53 at Ser15 which is mediated by the ATM and ATR protein kinases, and has long been considered to be an initiating and nucleating event in p53 activation [159]. Phosphorylation of Ser15 occurs within the first 30 min of a DNA damage stimulus. Although ATM activation can be transient (lasting only a few hours), subsequent slower activation of ATR occurs, possibly through the occurrence of single-stranded stretches of DNA generated by the repair mechanisms, thus providing a continuity of Ser15 phosphorylation that can endure for several hours after the initial stimulus.

Initiating a series of sequential events (Figure 6), phosphorylation of Ser15 generates a recognition determinant that allows CK1 to phosphorylate Thr18. Thr18 phosphorylation can potently contribute to uncoupling p53 from degradation [160166], most likely on the basis of electrostatic repulsion between the phosphorylated residue and several nearby acidic and aromatic residues at the surface of the MDM2 domain [167,168] (Figure 7A; a comparison with the p53–MDM4 structure suggests that similar events may regulate this interaction, see Figure 7B). Additionally, Brownian dynamics simulations suggest that phosphorylation of Thr18 and Ser20 can hinder p53 from interacting optimally with MDM2 well before reaching the p53-binding site [169]. An added advantage provided by Ser15 phosphorylation is that, by masking a nuclear export signal, it contributes to retaining p53 within the nucleus [170]. Phosphorylation of Ser20 by CHK2, which itself is phosphorylated and activated by ATM, contributes to uncoupling the p53–MDM2 interaction, especially in combination with Ser15 and Thr18 phosphorylation [160,164,166,171,172]. Notably, a S20A substitution mutant of p53 is intensely sensitive to degradation by MDM2 [171], underpinning the value of this residue in contributing to the regulation of p53 stability. The modification of these residues was originally thought to be the underlying mechanism of the DNA-damage-dependent induction (i.e. increased levels) of p53. However, the potent in vivo contribution of Mdm2-Ser394 phosphorylation towards p53 induction, established from the analysis of the Mdm2S394A/S394A mice (above), when compared with the subtle effects of p53S18A,S23A/S18A,S23A knockin mice on p53 levels [173] provides powerful evidence for the respective roles of these different sites in vivo (see below; Ser18 and Ser23 are equivalent to the Ser15 and Ser20 sites respectively of human p53).

Cascade of PTMs of p53 following Ser15 phosphorylation

Figure 6
Cascade of PTMs of p53 following Ser15 phosphorylation

The cascade of events following ATM/ATR activation is essentially as follows: (1) Ser15 is a target for phosphorylation by several reported protein kinases, mainly ATM and ATR. (2) pSer15 acts as a recognition determinant for protein kinase CK1 which subsequently phosphorylates Thr18. (3) ATM phosphorylates and activates CHK2 which then phosphorylates Ser20 and other key residues towards the C-terminus of p53. (4) Phosphorylation of N-terminal residues, mainly Thr18 and Ser20, contributes to uncoupling of the p53–MDM2 interaction and promotes interaction with p300 and CBP. Ser20 phosphorylation can also stimulate recruitment of hMOZ. (5) The substitution of histone acetyltransferases (HATs) for MDM2 diminishes ubiquitylation of lysine residues and promotes their acetylation. Importantly, the recruitment of these enzymes also leads to acetylation of histones in the chromatin immediately surrounding the p53-binding sites in promoters. (6) Additional phosphorylation events at Ser33 and Ser37 stimulate interaction with HATs including PCAF which acetylates Lys320, whereas recruitment of hMOZ leads to Lys120 acetylation. (8) Phosphorylation of Ser46 contributes to p53 activation, e.g. through recruitment of p300–p53 to apoptotic promoters. (9) ATM can impact on the modification of other sites such as Ser6 and Ser9. These are reported to be targeted by CK1 isoforms, but it is not clear whether ATM/ATR can regulate this enzyme directly. Abbreviations as in Figures 2 and 4, Table 1 and the text.

Figure 6
Cascade of PTMs of p53 following Ser15 phosphorylation

The cascade of events following ATM/ATR activation is essentially as follows: (1) Ser15 is a target for phosphorylation by several reported protein kinases, mainly ATM and ATR. (2) pSer15 acts as a recognition determinant for protein kinase CK1 which subsequently phosphorylates Thr18. (3) ATM phosphorylates and activates CHK2 which then phosphorylates Ser20 and other key residues towards the C-terminus of p53. (4) Phosphorylation of N-terminal residues, mainly Thr18 and Ser20, contributes to uncoupling of the p53–MDM2 interaction and promotes interaction with p300 and CBP. Ser20 phosphorylation can also stimulate recruitment of hMOZ. (5) The substitution of histone acetyltransferases (HATs) for MDM2 diminishes ubiquitylation of lysine residues and promotes their acetylation. Importantly, the recruitment of these enzymes also leads to acetylation of histones in the chromatin immediately surrounding the p53-binding sites in promoters. (6) Additional phosphorylation events at Ser33 and Ser37 stimulate interaction with HATs including PCAF which acetylates Lys320, whereas recruitment of hMOZ leads to Lys120 acetylation. (8) Phosphorylation of Ser46 contributes to p53 activation, e.g. through recruitment of p300–p53 to apoptotic promoters. (9) ATM can impact on the modification of other sites such as Ser6 and Ser9. These are reported to be targeted by CK1 isoforms, but it is not clear whether ATM/ATR can regulate this enzyme directly. Abbreviations as in Figures 2 and 4, Table 1 and the text.

Location of key phosphorylation sites in the N-terminus of p53 within the context of p53–MDM2 and p53–p300 associations

Figure 7
Location of key phosphorylation sites in the N-terminus of p53 within the context of p53–MDM2 and p53–p300 associations

The structures of the N-terminus of human p53 (blue) in complex with the N-terminus of MDM2 (grey, A), the N-terminus of MDM4 (grey, B) and the TAZ2 domain of human p300 (light blue, panel C) are shown. In (A) and (B), there are two views: termed ‘top view’ and, following rotation of approximately 90° around the horizontal plane, ‘side view’. The positions of serine (red) and threonine (magenta) phosphorylation sites are shown. Structural data are taken from the PDB (codes 3DAB and 1YCR respectively). In (C), there are four views, following rotation of approximately 90° around a vertical axis. The positions of serine (red) and threonine (magenta) phosphorylation sites are shown. Structural data are taken from the PDB (code 2K8F).

Figure 7
Location of key phosphorylation sites in the N-terminus of p53 within the context of p53–MDM2 and p53–p300 associations

The structures of the N-terminus of human p53 (blue) in complex with the N-terminus of MDM2 (grey, A), the N-terminus of MDM4 (grey, B) and the TAZ2 domain of human p300 (light blue, panel C) are shown. In (A) and (B), there are two views: termed ‘top view’ and, following rotation of approximately 90° around the horizontal plane, ‘side view’. The positions of serine (red) and threonine (magenta) phosphorylation sites are shown. Structural data are taken from the PDB (codes 3DAB and 1YCR respectively). In (C), there are four views, following rotation of approximately 90° around a vertical axis. The positions of serine (red) and threonine (magenta) phosphorylation sites are shown. Structural data are taken from the PDB (code 2K8F).

Several other residues in the TAD1 region of p53 undergo DNA-damage-induced phosphorylation, including Ser6, Ser9, Ser33 and Ser37 [174] with phosphorylation of Ser9 and Ser33 showing a dependence on ATM [159]. Modification of these residues does not appear to affect the interaction of p53 with MDM2 [164,166].

PTM has a dual role, acting as a switch between p53 degradation and activation, and as a rheostat that modulates the levels of activity

PTM of p53 not only down-regulates MDM2 binding, but also stimulates association with key transcription factors. It can thus act as a switch between degradation and activation. Over and above this mechanism, phosphorylation of individual residues in the N-terminus (TAD1) of p53 stimulates, to various extents, interaction with p53-binding sites in the p300 and CBP transcriptional co-activator proteins (reviewed in detail in [105,108]). Biophysical analyses measuring the interaction of p53 representative peptides with p300 indicate that the most potent event is phosphorylation of Thr18. Moreover, these analyses show that di- or multiple phosphorylation events can act co-operatively, leading to stimulation of the interaction as much as 80-fold. Analysis of TAD1 of p53 in association with the TAZ2 domain of p300 provides an explanation as to how this may occur. (Note that p300 contains four independent, but strikingly similar, domains termed TAZ1, KIX, TAZ2 and IBID respectively, to which p53 can bind. In the ‘wrap-around’ model proposed by Fersht and colleagues [175], one monomer of p300 interacts simultaneously with each of the subunits in tetrameric p53.) In the context of the p53–p300 interaction, Ser15 and Thr18 lie at the interface with p300 (Figure 7C; details given in [162]) with the consequence that their phosphorylation will strengthen contact through increased electrostatic bond formation with critical residues in p300. Combinations of these and other phosphorylation events in TAD1 of p53 appear to act co-operatively in stimulating p300/CBP binding in a graded or incremental manner [163,164,166,176,177]. If these in vitro analyses hold true for p53 in a cellular context, they may function in the capacity of a rheostat that allows p53 activity to be fine-tuned. As with Ser15 and Thr18, the structure of the N-terminus of p53 in association with the p300 TAZ2 domain [162] reveals the close proximity of other phosphorylation sites to the p300 surface where their modification status may affect the strength of association [105] (Figure 7C).

The central role of acetylation in activating p53 and mediating its downstream effects

Acetylation of key lysine residues in p53 itself [178], and of histone proteins in the neighbouring chromatin of p53-binding sites at promoters [179], are critical events in the activation mechanism. Acetylation is dependent on the recruitment of histone acetyltransferases, such as CBP and p300, which, as described above, can be regulated through sequential and co-operative N-terminal phosphorylation events. The acetylation of p53 has recently been described in detail in an excellent review [104] and therefore only the relevant and salient points are discussed here.

Briefly, six lysine residues in the C-terminus of p53, Lys370, Lys372, Lys373, Lys381, Lys382 and Lys386, are acetylated through the action of CBP and p300. These are the same lysine residues that are major targets for ubiquitylation by MDM2. Consequently, acetylation (or methylation) of these residues is mutually exclusive with their ubiquitylation and can therefore serve as a mechanism that opposes the down-regulation of p53 by MDM2 [180]. Acetylation of p53 can also: (i) block recruitment of MDM2 and MDM4 to p53 to form repressive complexes on promoters; (ii) facilitate the further recruitment of promoter-specific transcription factors towards further activating transcription [178,181]; and (iii) improve the ability of p53 to bind to certain target genes (see [104] and references therein).

There are three other important sites of acetylation in p53. Acetylation of Lys320 in the tetramerization domain by PCAF [182] promotes transactivation of cell-cycle-arrest genes by p53 and, consequently, favours growth arrest over cell death [183]. This role is supported by the finding that K317R knockin mice, which cannot be acetylated at this position, show enhanced p53-dependent apoptosis following irradiation [184] (Lys317 is the murine equivalent of human Lys320). Acetylation of Lys120 in the DNA-binding domain by TIP60 (60 kDa tail-interacting protein) (KAT5)/hMOF [human orthologue of MOF (males absent on the first)] (MYST1/KAT8) occurs rapidly following a DNA damage stimulus. In contrast with Lys320, this modification is indispensable for the activation of p53 target genes encoding apoptosis-associated proteins, but has negligible influence on the expression of cell-cycle-arrest genes [185,186]. Lys120 acetylation may also influence the transcription-independent apoptotic function of p53. The third site, Lys164, is also located in the core domain, is a substrate for p300 and CBP, and is needed for the activation of most p53 target genes [178].

Investigation of the function of the cluster of C-terminal lysine residues in vivo using knockin cells and mouse models in which six or seven of these residues are replaced by arginine (p536KR and p537KR) revealed only mild phenotypes and generally showed no major differences in growth arrest, apoptosis or tumour suppression [74,75]. These conclusions are consistent with the idea that modification of the C-terminal lysine residues might be compensated for by other sites, especially given that p536KR and p537KR can be ubiquitylated on other lysine residues. On the other hand, collective arginine substitution of Lys120, Lys164 and the C-terminal lysine residues (p538KR) significantly impairs the ability of p53 to stimulate expression of genes required for apoptosis or growth arrest yet can still drive expression of MDM2, at least in cultured cells [178]. These findings provide support for the idea that acetylation is required to mediate the expression of at least some p53-downstream genes and, accordingly, drive p53 biological function. They can thus explain how initiating events such as DNA damage can lead to stimulation of p53 function and outcome.

More recently, the analysis of Ser15 phosphorylation has been extended towards understanding its role in relaxing chromatin within a well-characterized p53-responsive promoter (the CDKN1A promoter, encoding p21) [131]. Here, it has been shown that p53 harbouring an alanine substitution at residue 15 (S15A) is unable to promote neighbouring histone acetylation and transcriptional activation. Consistent with the need for phosphorylation, substituting an aspartate (phosphomimetic) residue for this position could restore histone acetylation and transactivation. This study thus provided a clear mechanistic link between a DNA-damage stimulus, phosphorylation of a site with an established critical role in recruiting transcriptional co-activators and local chromatin relaxation, leading to stimulation of p21 expression. Incidentally, Ser15 phosphorylation may also be required for the p53-dependent repression of at least some genes. For example, wild-type p53, but not a S15A mutant, inhibits the expression of Bcl-3, a member of the IκB (inhibitor of NF-κB) family that functions as a transcriptional co-activator for p52 NF-κB [187]. It will be interesting to know whether other targets for p53 repression are also regulated through post-translational mechanisms.

Many other sites of modification in p53 are responsive to DNA damage

In addition to the major PTMs on p53 described above that occur following a DNA-damage stimulus, a range of other modifications have been reported that include phosphorylation, acetylation, SUMOylation, NEDDylation and glycosylation events. These have been described and discussed in significant detail in other reviews [34,104,105,188190]. p53 is also regulated by a complex series of methylation events on the various C-terminal lysine residues discussed above that undergo ubiquitylation, acetylation and other modifications. Methylation can be DNA-damage-responsive and activating: for example, SET7/9-mediated methylation of Lys372 can promote acetylation of neighbouring lysine residues, leading to stabilization and activation of p53. Methylation can also act repressively by blocking acetylation in the absence of DNA damage and keeping p53 in a less active state. Detailed analysis of the methylation of p53 has been reviewed in depth elsewhere [191,192].

For the purpose of the present review, a list of well characterized DNA-damage-responsive PTMs of p53 is presented in Table 1 (together with selected relevant references where they are not cited in the text). These modifications are highlighted to underscore an important underlying principle in p53 regulation, i.e. that there are variations in the choice and magnitude of PTMs of p53 depending on the nature of the inducing stimulus (which reflects the type of DNA damage acquired), and the intensity and duration of the stimulus (see, e.g., [193,194]. The ability of these modifications, in the relevant combinations, to modulate interactions, govern promoter selectivity and influence the biological outcome of inducing p53, is thought to underpin the ‘barcode hypothesis’: an epigenetic-like code in which the p53 response is tailored, or fine-tuned, appropriately to match and respond to the nature and intensity of the activating stress [195,196].

Table 1
Relationship of phosphorylation sites in p53 to the DNA-damage response

53BP1, p53-binding protein 1; DYRK2, dual-specificity tyrosine-phosphorylation-regulated kinase 2; HIP2K, homeodomain-interacting protein kinase 2; PP2A, protein phosphatase 2A; TFIIH, transcription factor IIH.

Site Modifying enzyme(s) p53 domain Function(s) Selected reference(s) 
 Phosphorylation    
Ser6 CK1 TAD1 Increased binding to SMAD proteins [235,237
Ser9 CK1, ATM TAD1 Increased binding to SMAD proteins [159,194,235,237
Ser15 ATM, ATR, DNA-PK and others TAD1 Nucleation of subsequent PTMs; recruitment of CBP/p300; relaxation of chromatin in vicinity of p53-binding sites See the text 
Thr18 CK1 TAD1 Uncoupling MDM2; recruitment of CBP/p300 See the text 
Ser20 CHK2 TAD1 Uncoupling MDM2; recruitment of CBP/p300 See the text 
Ser33 p38 TAD1 Activation in response to UV and osmotic shock [256,257
Ser37 ATR TAD1 Uncoupling of p53 from replication protein A [258
Ser46 HIPK2, DYRK2, protein kinase Cδ, AMPKα TAD2 Promoting apoptosis through selective gene expression; induced at high levels of DNA damage; increased binding of TAD2 to p62 subunit of TFIIH [259269
Ser55 TAF1 TAD2 p53 turnover, cell cycle progression, interaction with CRM1 and nuclear export; constitutively phosphorylated in unstressed cells; dephosphorylated in response to DNA damage by PP2A; increased binding of TAD2 to p62 subunit of TFIIH [260,261,270272
Thr81  Proline-rich Recruitment of PIN1 peptidyl isomerase to promote conformational change; generation of binding site for CHK2 [273
Ser155 COP9-signalosome (CSN) Core Targeting of p53 for degradation [274
Ser215 Aurora A Core Blocking of site-specific DNA binding and promoting turnover [275
Ser269 Not known Core (ubiqutylation signal) Inactivation of p53, may induce a conformational shift characteristic of mutant p53 proteins [276,277
Ser315 CDK2, Aurora A Beyond C-terminal end of core Blocking site-specific DNA binding and promoting turnover; stimulating transactivation function; responsive to endoplasmic reticulum (protein unfolding) stress [278281
Ser376 GSK3β C-terminal regulatory region Constitutively phosphorylated in unstressed cells; DNA damage promotes dephosphorylation and permits 14-3-3 binding leading to increased site-specific DNA binding [280,282
Ser378 Not known C-terminal regulatory region Constitutively phosphorylated in unstressed cells [282
Ser392 CK2 C-terminus Promotion of tetramerization and site-specific DNA binding in vitro; required for anti-proliferative activity; protective against chemically induced bladders carcinomas and UV-induced skin tumours [201,283289
 Acetylation    
Lys120 hMOF, TIP60 Core Required for apoptosis through selective expression of apoptosis-related genes See the text 
Lys164 p300, CBP Core Required for growth arrest and apoptosis See the text 
Lys320 PCAF Beyond C-terminal end of core Increases p53 DNA binding; regulates transcriptional activity See the text 
Lys370, Lys372, Lys373, Lys381, Lys382, Lys386 p300, CBP C-terminal regulatory region Enhances sequence-specific DNA binding; promotes recruitment of p300/CBP and other transcription factors; mutually exclusive with ubiquitylation of same residues; contributes to p53 stability See the text 
 Methylation    
Lys370 SMYD2 C-terminal regulatory region Repression (inhibition of promoter association) [290
Lys372 SET7/9 C-terminal regulatory region DNA-damage-induced; promotes acetylation (through recruitment of TIP60) and stabilization [291,292
Lys382 SET8/PR-Set7 C-terminal regulatory region Mono-methylation promotes interaction with L3MBTL1 and suppresses p53-dependent transcription; DNA damage promotes dimethylation, interaction with Tudor domain of 53BP1, and p53 stabilization [293295
Site Modifying enzyme(s) p53 domain Function(s) Selected reference(s) 
 Phosphorylation    
Ser6 CK1 TAD1 Increased binding to SMAD proteins [235,237
Ser9 CK1, ATM TAD1 Increased binding to SMAD proteins [159,194,235,237
Ser15 ATM, ATR, DNA-PK and others TAD1 Nucleation of subsequent PTMs; recruitment of CBP/p300; relaxation of chromatin in vicinity of p53-binding sites See the text 
Thr18 CK1 TAD1 Uncoupling MDM2; recruitment of CBP/p300 See the text 
Ser20 CHK2 TAD1 Uncoupling MDM2; recruitment of CBP/p300 See the text 
Ser33 p38 TAD1 Activation in response to UV and osmotic shock [256,257
Ser37 ATR TAD1 Uncoupling of p53 from replication protein A [258
Ser46 HIPK2, DYRK2, protein kinase Cδ, AMPKα TAD2 Promoting apoptosis through selective gene expression; induced at high levels of DNA damage; increased binding of TAD2 to p62 subunit of TFIIH [259269
Ser55 TAF1 TAD2 p53 turnover, cell cycle progression, interaction with CRM1 and nuclear export; constitutively phosphorylated in unstressed cells; dephosphorylated in response to DNA damage by PP2A; increased binding of TAD2 to p62 subunit of TFIIH [260,261,270272
Thr81  Proline-rich Recruitment of PIN1 peptidyl isomerase to promote conformational change; generation of binding site for CHK2 [273
Ser155 COP9-signalosome (CSN) Core Targeting of p53 for degradation [274
Ser215 Aurora A Core Blocking of site-specific DNA binding and promoting turnover [275
Ser269 Not known Core (ubiqutylation signal) Inactivation of p53, may induce a conformational shift characteristic of mutant p53 proteins [276,277
Ser315 CDK2, Aurora A Beyond C-terminal end of core Blocking site-specific DNA binding and promoting turnover; stimulating transactivation function; responsive to endoplasmic reticulum (protein unfolding) stress [278281
Ser376 GSK3β C-terminal regulatory region Constitutively phosphorylated in unstressed cells; DNA damage promotes dephosphorylation and permits 14-3-3 binding leading to increased site-specific DNA binding [280,282
Ser378 Not known C-terminal regulatory region Constitutively phosphorylated in unstressed cells [282
Ser392 CK2 C-terminus Promotion of tetramerization and site-specific DNA binding in vitro; required for anti-proliferative activity; protective against chemically induced bladders carcinomas and UV-induced skin tumours [201,283289
 Acetylation    
Lys120 hMOF, TIP60 Core Required for apoptosis through selective expression of apoptosis-related genes See the text 
Lys164 p300, CBP Core Required for growth arrest and apoptosis See the text 
Lys320 PCAF Beyond C-terminal end of core Increases p53 DNA binding; regulates transcriptional activity See the text 
Lys370, Lys372, Lys373, Lys381, Lys382, Lys386 p300, CBP C-terminal regulatory region Enhances sequence-specific DNA binding; promotes recruitment of p300/CBP and other transcription factors; mutually exclusive with ubiquitylation of same residues; contributes to p53 stability See the text 
 Methylation    
Lys370 SMYD2 C-terminal regulatory region Repression (inhibition of promoter association) [290
Lys372 SET7/9 C-terminal regulatory region DNA-damage-induced; promotes acetylation (through recruitment of TIP60) and stabilization [291,292
Lys382 SET8/PR-Set7 C-terminal regulatory region Mono-methylation promotes interaction with L3MBTL1 and suppresses p53-dependent transcription; DNA damage promotes dimethylation, interaction with Tudor domain of 53BP1, and p53 stabilization [293295

Attenuation of DNA damage-dependent induction of p53

p53 is involved in multiple positive- and negative-feedback loops that maintain its levels and activity [102], some of which may contribute to restoring p53 homoeostasis following termination of a stress stimulus. The role of MDM2 in attenuating the p53 response is well established [102] and was demonstrated strikingly by time-lapse experiments in individual cells expressing colour-labelled p53 and MDM2 proteins: in these analyses, DNA damage-induced p53–CFP led to increased MDM2–YFP with subsequent disappearance of p53–CFP and so on [197]. The contribution of the p53–MDM2 loop in vivo is supported by studies of mutant p53 expression in mice and in zebrafish, before and after treatment with IR [78,79]. These studies showed that, as with wild-type p53, mutant p53 is maintained at low levels in normal cells by MDM2 expressed from the p53-independent promoter, one of two promoters in the MDM2 gene. Following a stimulus, however, mutant p53 is unable to induce the higher levels of MDM2 from the alternative p53-sensitive promoter in the MDM2 gene that is required to attenuate the response. Thus, upon DNA damage, the levels of mutant p53 significantly exceed wild-type p53 levels, induced under identical conditions, and persist for a much longer period of time [78,79].

The WIP1 protein phosphatase also operates within a feedback loop with p53. WIP1 dephosphorylates a number of DNA-damage-signalling proteins in vivo [198], including phospho-Ser395 in MDM2, leading to p53 destabilization, and phospho-Ser15 in p53 itself [199]. Indirectly, WIP1 can reduce the phosphorylation of other key p53 residues such as Ser20, Ser33 and Ser46 through its ability to down-regulate the pathways that modify these sites [200]. The finding that overexpression, or GOF mutation, of PPMD1 (encoding WIP1) is frequently observed in human cancers fits with the idea that down-regulation of p53 signalling, including the dephosphorylation of p53 and/or its partners and upstream modifiers, can contribute to tumour development.

These examples provide evidence of mechanisms that operate to reverse the activating events discussed above, leading to attenuation of the p53 response and the restoration of homoeostasis.

An overarching fundamental role of Ser15 phosphorylation

Although many PTMs on p53 itself have been viewed primarily in the context of the DNA-damage response, recent evidence suggests that Ser15 phosphorylation, the key event in the activation of p53 in response to DNA damage, may have a much broader or even universal role in activating p53 than had previously been anticipated. First, basal (uninduced) levels of p53, and p53 that is induced simply through inhibiting MDM2 pharmacologically, show detectable levels of Ser15 phosphorylation [131,201]. Therefore, although DNA damage can robustly induce Ser15 phosphorylation, other mechanisms operate that permit this modification to occur under different circumstances, albeit at relatively lower levels.

Secondly, replacing Ser15 in human p53 with alanine, which cannot be phosphorylated, greatly impairs the transcriptional activity and biological response of p53 (as determined by measuring growth arrest) when it is expressed at levels closely matching those of endogenous p53 in commonly used cell lines [131]. Replacing with aspartate, a phosphomimetic, maintains p53 transcriptional and biological functions under these circumstances. The recruitment of histone acetyltransferase activity (e.g. p300) by p53 leads to the acetylation of p53-bound nucleosomes in the p21 promoter [179]. The inability to phosphorylate p53 at Ser15 impedes this recruitment and thus impairs histone acetylation and chromatin relaxation at p53-responsive sites in target promoters [131]. Moreover, p53 is found to be pre-loaded on certain promoters in unstressed cells (e.g. the p21 promoter), where it is poised ready to commit to transcription following an appropriate stimulus [202]. Although occupancy of this site before a stimulus is independent of Ser15 phosphorylation, reloading of S15A-p53 post-induction is significantly decreased compared with wild-type p53, even when the stimulus is the MDM2 inhibitor Nutlin [131]. Taken together, these observations make a strong case that the integrity of this modification robustly underpins the ability of p53 to operate at full strength, irrespective of the initiating stimulus.

These conclusions suggest that regulation of p53 activity through PTM is an integral part of its regulation in a wider context than DNA damage alone. Additionally, whereas the anti-repression model views the induction of p53 essentially as the release of active p53 from MDM2/MDM4 repression, the impact of important modifications such as Ser15 phosphorylation, which may actually be masked by MDM2/MDM4 binding, cannot be ignored.

Other areas of cross-talk between the different p53-activating pathways

In addition to Ser15, phosphorylation of other residues (including Ser33, Ser46 and Ser392) has been detected in p53 that is induced through the ARF pathway and through Nutlin treatment [201]. Moreover, there is now good evidence that the ARF/oncogene pathway activates p53 Lys120 acetylation within the DNA-binding domain [203] and can stimulate the acetylation of Lys382 at the C-terminus [204]. Similarly, acetylation of p53 occurs in response to various stress-inducing agents that do not induce DNA damage, such as hypoxia, anti-metabolites and a nuclear export inhibitor [205]. These findings indicate that PTMs do occur independently of DNA damage and are therefore likely to have functional relevance beyond the DNA-damage response. Regarding the pathways themselves, functional interaction between ATM and ARF has also been demonstrated in cultured cells and in human cancers consistent with an ongoing cross-talk [206]. Moreover, ARF can activate ATR and CHK1, two DNA damage-responsive protein kinases that play a pivotal role in the p53 response to DNA damage, both leading to increased p53 phosphorylation [207]. Through Chk1 activation and RelA phosphorylation, ARF additionally down-regulates the ability of the NF-κB pathway to oppose p53-dependent apoptosis [207]. These links identify a clear interdependence among the various mechanisms through which the p53 response is activated.

THE RELEVANCE AND CONTEXT DEPENDENCE OF DIFFERENT p53-ACTIVATING PATHWAYS TO TUMOUR SUPPRESSION

The character of every act depends upon the circumstances in which it is done.”–Oliver Wendell Holmes, Jr

The mechanism of induction/activation of p53, and the duration of the response, are now considered to be key factors in determining whether p53 tumour-suppressor activity is activated. Developing cancer cells undergo numerous stresses, including hypoxia, nutrient limitation, hyperproliferative signalling (activated oncogenes) and persistent DNA damage (e.g. arising from inappropriate activation of origins of replication [208,209]), any of which could activate p53. However, compelling evidence from mouse models indicates that, whereas the acute DNA-damage response is likely to be dispensable for tumour suppression, the ARF (hyperproliferative signalling) pathway has a critical role in this process. For example, ARF inactivation during lymphomagenesis in an Eμ-Myc model for Burkitt's lymphoma is sufficient to eliminate p53-dependent tumour suppression [53]. Moreover, two independent studies [210,211], each using engineered mice in which p53 expression is absent but can be restored upon administration of an appropriate drug, demonstrate that the acute p53-mediated response to DNA damage fails to trigger the p53 tumour-suppression function. In contrast, restoration of p53 at various intervals after attenuation of the DNA-damage response is sufficient to protect against lymphoma development and in a manner that can be eliminated in an ARF-null background. Consistent with these studies, p53 ‘super’ mice, which have an extra transgenic copy of the intact Trp53 gene, and which show an increased response to genotoxic agents, are unable to suppress tumour formation when crossed into an ARF-null background [212]. Furthermore, oncogene-induced senescence and tumour suppression in mice can occur in the absence of a detectable DNA damage response and in an Atm-null background [213]. These studies not only favour the idea that the ARF pathway, and not the acute DNA-damage response, is crucial for preventing the development of cancer (at least in the mouse models tested), but also call into question what relevance, if any, many of the DNA-damage-induced modifications discussed above have in suppressing cancers. This is underscored by the finding that knockin mice expressing the transcriptionally defective L25Q/W26S substitution mutant of p53 (p53L25Q/W26S, which lies within TAD1) fail to transactivate many well characterized pro-arrest and pro-apoptotic genes and cannot induce growth arrest or apoptosis, yet effectively suppress the development of various tumour types driven by different activated oncogenes [55]. Given that many of the DNA damage-induced phosphorylation sites are located within this region, it seems unlikely that they are generally or broadly required for tumour suppression.

THE BIOLOGICAL CONTEXT OF THE POST-TRANSLATIONAL MECHANISMS THAT REGULATE p53

For me context is the key; from that comes the understanding of everything.”–Kenneth Noland

Over the last few years, there has been a major effort towards understanding mechanisms of p53 regulation in a biological context, including the role of PTMs, using genetically engineered mouse models (these have been reviewed in detail elsewhere [88,105,189,214216]). It is valuable to reconsider a selection of these in the light of recent knowledge acquired from biochemical and cellular studies (discussed above) with a view towards understanding relationships between the molecular mechanisms and the biological and/or pathological relevance or consequences of these events. For example, in mice homozygously expressing an S18A substitution mutant (p53S18A/S18A; equivalent to Ser15 in human p53), protein stabilization is not significantly affected in response to DNA damage [217,218]. This observation fits with the recent demonstration that DNA damage induces p53 mainly through controlling events at MDM2 [134]. However, C-terminal acetylation is defective in the p53S18A/S18A mice, and the ability to transactivate various genes and induce apoptosis is compromised. This is consistent with recent work highlighting a key role for Ser15 in transcription and promoter relaxation [131]. Curiously, these mice are not prone to spontaneous tumorigenesis and this again fits, in a general sense, with recent findings that functional and regulatory events within TAD1 of p53 are not essential for tumour suppression [55].

p53S23A/S23A (equivalent to Ser20 in human p53) mice have also been examined. Here, cell-type-specific differences are apparent such that IR-induced increases in p53 protein levels and apoptosis are modestly impaired in thymocytes but are unaffected in MEFs [173,219]. Thus our molecular understanding of the role of key modifications seems applicable to some cell types, but only in a contributory sense, and redundant or not relevant in others. Additionally, p53S23A/S23A mice develop B-cell lymphomas, highlighting a contributory role for this phosphorylation site to tumour suppression in at least one tumour type. Interestingly, mice in which both of these phosphorylation sites have been substituted (p53S18A,S23A/S18A,S23A) support, to some extent, the biochemical evidence that these two phosphorylation sites co-operate in regulating p53 function. Thus thymocytes from the p53S18A,S23A/S18A,S23A mice show significantly impaired p53 stabilization, transactivation capacity and apoptosis in response to irradiation, although, again consistent with cell type differences, these impairments were less evident in MEFs [220]. Additionally, the p53S18A,S23A/S18A,S23A mice are cancer-prone, but show a significantly slower onset compared with p53-null mice, and develop a spectrum of malignancies that is distinct from p53-null mice. These findings highlight a meaningful contribution of these phosphorylation sites to tumour suppression but, most likely, in a cell-type-dependent fashion. Interestingly, p53T21D,S23D/− mice, which express aspartate residues (phosphomimetics) at two key N-terminal phosphorylation sites have constitutively active p53, and show depletion of stem cell populations and signs of premature aging that can be rescued by deletion of the Puma gene [221] (Puma is a pro-apoptotic p53 target gene). It is not clear what impact these substitutions have on cancer susceptibility. However, the results from studying these mice are consistent with new findings (discussed above) that inappropriate activation of p53 is linked to various non-cancer pathologies. Thus, whereas PTMs stimulated by DNA damage or other signalling pathways may contribute in part, or not at all, to tumour suppression, and in a cell- or tissue-type manner, the biological and pathological relevance of these modifications may lie in other aspects of p53 function.

SIGNALLING TO GAIN-OF-FUNCTION MUTANT p53 PROTEINS AND THE RAMIFICATIONS FOR CANCER TREATMENT

What is PARADOXICAL REACTION?……with regard to pharmacology, a drug response which contradicts the predicted effect.”–Psychology Dictionary

As discussed above, many missense mutant p53 proteins acquire new oncogenic functions that provide a selective advantage to cancer cells. Most, but not all, of these mutations occur in the core domain, leading to loss of site-specific DNA binding and wild-type transcription function. However, other domains may be unaffected by mutation and still be able to mediate the interactions through which they normally function. It follows that these domains may also still be able to act as targets for PTM and/or protein–protein association events, with the outcome that their interactions can still take place and be regulated.

There is good evidence that mutant p53 proteins are indeed post-translationally modified (this has been discussed in detail elsewhere [190]). As far back as 1993, Ullrich et al. [222] showed that Ser15 and Ser392 were phosphorylated in two mutant p53 proteins. Subsequent analyses over the last two decades by various groups has confirmed that many of the phosphorylation sites characteristic of wild-type p53 are also phosphorylated in a variety of mutant p53 proteins, both in cell lines derived from human tumours and, indeed, in the cancers themselves [223233]. Although various patterns of modification have been observed, possibly reflecting different conditions (such as different cell lines, specific p53 mutations, types and stage of tumour), these findings suggest very strongly that the stress stimuli and/or signalling pathways that modulate wild-type p53 can also target mutant p53 proteins, and raise the possibility that activation of appropriate signalling pathways could have a measureable impact on mutant p53 GOF. Indeed, some studies have already confirmed that phosphorylation of key residues in mutant p53 can be stimulated in response to different stresses [231,233], whereas others have demonstrated that mutant p53 phosphorylation can promote its subsequent acetylation and enhance its interaction with transcriptional proteins such as p300 [232]. A more detailed example is the phosphorylation of Ser6 and Ser9 by the protein kinase CK1 family members CK1δ and CK1ε in response to signalling through the MAPK (mitogen-activated protein kinase) pathway. These modifications occur both in physiological and pathological contexts, and permit the interaction of p53 with TGFβ (transforming growth factor β)-activated SMAD2 proteins [234237]. Phosphorylation of the p53R280K mutant in response to activated Ras signalling leads to the formation of a mutant p53–SMAD complex that recruits the p53 family member p63, and inhibits its anti-metastatic activity [234]. In this example, phosphorylation of a GOF mutant p53 protein can thus contribute to the well-characterized switching of TGFβ action from that of a tumour suppressor to a potent pro-metastatic factor. The possibility that PTMs may actually promote mutant p53 activities might also help to explain why mutations in the TP53 gene encoding phosphorylation target residues are rarely, if ever, seen in human cancers as the fidelity of such residues may be required to underpin the potency of mutant p53 proteins.

Additionally, mutant p53, like wild-type p53, is inherently unstable in normal cells (mainly through MDM2-dependent ubiquitylation/proteasomal degradation), but becomes stabilized in cancer cells [78]. Stabilization of mutant p53 is largely responsible for GOF in mouse models [78] and anti-cancer drugs used in the clinic (such as the anthracycline doxorubicin) promote stabilization of mutant p53 in mouse models, leading to increased tumour burden and significantly earlier death [238]. Although there is evidence of the potential involvement of PTM in inducing and activating mutant p53 (e.g., see [239]), the mechanisms by which these events occur have not been explored in any depth. The knowledge that many clinical anti-cancer agents are DNA-damage-inducing compounds that stimulate phosphorylation and activation of wild-type p53 raises the possibility that similar events in mutant p53 may facilitate interactions that sequester important transcription factors needed for the expression of other key genes and/or stimulate expression of those genes for which mutant p53 plays a significant role [62]. Thus, given that standard chemotherapy-based treatments could actually promote disease in patients with relevant TP53 mutations, understanding these mechanisms is both urgent and important.

CONCLUSIONS AND PERSPECTIVES

And let us, ciphers to this great account, On your imaginary forces work.”–William Shakespeare (Henry V)

The p53 pathway is activated through various distinct mechanisms, largely according to the type of initiating stimulus. Although the key focal point of these activating mechanisms is the p53–MDM2 interaction, it is clear that p53 induction alone (i.e. increased p53 levels) does not always provide the most appropriate adaptive response. Accordingly, a variety of mechanisms operate to fine-tune the magnitude, duration and aptness of the response. These mechanisms are likely to carry different weight, depending on cell and/or tissue type, and can have a selective influence on the ability of p53 to suppress tumour formation. Moreover, although certain aspects of activating mechanisms had previously been considered to be confined to a particular pathway (e.g. p53 phosphorylation was considered to be selective for the DNA-damage response), it is now clear that there is a significant degree of mechanistic overlap between established routes of activation including the responses to DNA damage, hyperproliferation, nucleolar stress and pharmacological activation. This new perception raises a number of interesting issues for further consideration and exploration.

Do different p53-activating pathways have a differential requirement for PTMs?

It is now clear that PTMs such as phosphorylation and acetylation do occur on p53 in response to non-DNA-damage-induced signalling. However, it is also clear that the levels of modification may vary: for example, key phosphorylation events induced by DNA damage are significantly greater in magnitude than those produced by ARF or Nutlin treatment [131,201]. It is not clear why this is the case. One possible explanation is that phosphorylation contributes towards implementing the ‘barcode hypothesis’ and may thus shape the p53 response in an adaptive manner to respond appropriately to the type and intensity of the DNA damage and, possibly, to the needs of the individual cell type. Although such fine-tuning would provide a highly sensitive mechanism of managing the damage, it raises the question of why other types of stress do not elicit a similar extent of phosphorylation. It may be that the complexity of events involved in detecting damage to, and mediating repair of, DNA necessitate an equally complex surveillance from p53 in order to achieve the most appropriate outcome.

It is also possible that, in the DNA-damage response, p53 phosphorylation is required to co-operate with MDM2 phosphorylation to produce a full and robust induction. Such co-operation may not be required if MDM2 is inactivated through ARF or ribosomal protein binding. Recent evidence suggests that Ser15 phosphorylation plays a pivotal role in activating p53-dependent transcription and that the comparatively lower levels of Ser15 phosphorylation observed following a stimulus such as Nutlin treatment are sufficient for transcriptional activity. These apparently lower levels of phosphorylation may, of course, simply reflect a greater degree of turnover of the modification (i.e. phosphorylation/dephosphorylation) in non-DNA-damage-derived signalling. Alternatively, they may reflect the ‘rheostat’ idea of turning p53 activity up or down as is required. Thus it is possible that low levels of phosphorylation underpin transcriptional function, whereas higher levels are needed to maintain uncoupling from MDM2.

A third possibility is that, if PTMs occur at promoters by promoter-localized mechanisms (e.g. recruitment of signalling enzymes to specific promoters), this could also help to explain differences in modifications and levels. Such a mechanism would provide scope for very selective and stimulus-specific gene expression.

What do the various PTMs do in pathologies other than cancer?

One example given above (the p53T21D,S23D/− mouse) indicates that inappropriate modification of p53 may lead, or contribute, to some of the diseases mentioned at the start of the present review (e.g. neuropathies) where inappropriate activation of p53 is detrimental. It will therefore be interesting to have a more comprehensive understanding as to whether signalling pathways operating in these pathological conditions lead to changes in the p53 activation status. One potential means of addressing these issues would be to introduce modification site mutants of p53 into mouse models for these various diseases. Information gained from analyses of this type could identify pathways or signalling molecules that could be targeted therapeutically towards alleviating or ameliorating the conditions.

Do PTMs regulate p53 isoforms?

It is now clear that, in addition to the full-length p53 molecule, there are another possible 11 isoforms, many of which may contribute to regulating the p53 response and/or interacting with other p53 family members [40]. Given that these isoforms still retain many of the sites of PTM established in full-length p53, it is entirely possible that they are modified in a similar manner in isoforms and in response to similar stimuli. It will therefore be important to understand whether indeed these modifications occur and how they affect the outcome of isoform function.

Do PTMs regulate other functions of p53?

As discussed above, p53 is now known to play roles in many biological processes including fertility, stem cell renewal, the immune response, aging, pigmentation and metabolism. The question then arises as to how p53 levels and function are regulated in these various events. p53 can regulate proliferation through AMPK (AMP-regulated protein kinase) pathway-dependent Ser15 phosphorylation in response to changes in glucose levels [240]. Moreover, the ability of p53 to co-ordinately regulate the levels of key metabolic enzymes has acquired enormous significance, both from a physiological perspective and in the context of cancer suppression by opposing the Warburg effect (aerobic glycolysis in cancer cells) [17,241]. It is therefore quite striking that S18A mice show defects in glucose metabolism and acquire a degree of insulin insensitivity [242]. Furthermore, ATM, which is one of the protein kinases that phosphorylates this site, has also been implicated in areas such as metabolism and insulin sensitivity, oxidative stress, aging and mitosis [243247]. Targeting of p53 and its associated regulators by ATM may therefore extend beyond the boundaries of the DNA damage response and mediate sensitivity to, and surveillance of, other key biological processes.

p53 has also been long known to play a role in responding to cells exiting from aberrant mitoses without undergoing cell division, either by driving them into apoptosis or by arresting them in a G1-like state with 4N DNA [248]. Recent evidence has shown that the mechanism of p53 activation involves loss of telomere protection, through the removal of TRF2, leading to activation of p53 through the DNA-damage response [249252]. These new findings again provide examples of novel functions of p53 that are regulated through established pathways and post-translational events.

Clearly, the regulation of the p53 response is a fascinating area of molecular biology that offers enormous scope for further exploration in terms of mechanistic events, cross-talk between different pathways, involvement in regulating novel p53 functions, contributing to the development of cancer and other, unrelated, diseases, and offering scope for novel approaches towards alleviating these conditions. The next few years should provide us with some promising developments in this area.

Abbreviations

     
  • AMPK

    AMP-regulated protein kinase

  •  
  • ARF

    alternative reading frame

  •  
  • ATM

    ataxia telangiectasia mutated

  •  
  • ATR

    ataxia telangiectasia- and Rad3-related

  •  
  • BAX

    Bcl-2-associated X protein

  •  
  • CBP

    cAMP-response-element-binding protein-binding protein

  •  
  • CDK

    cyclin-dependent kinase

  •  
  • CHK

    checkpoint kinase

  •  
  • DNA-PK

    double-stranded DNA-activated protein kinase

  •  
  • GOF

    gain-of-function

  •  
  • GSK3β

    glycogen synthase kinase 3β

  •  
  • HAUSP

    herpesvirus-associated ubiquitin-specific protease

  •  
  • hMOF

    human orthologue of MOF (males absent on the first)

  •  
  • IR

    ionizing radiation

  •  
  • KAT

    lysine acetyltransferase

  •  
  • MDM

    murine double minute

  •  
  • MEF

    mouse embryonic fibroblast

  •  
  • α-MSH

    α-melanocyte-stimulating hormone

  •  
  • NF-κB

    nuclear factor κB

  •  
  • PCAF

    p300/CBP-associated factor

  •  
  • PPM

    Mg2+/Mn2+-dependent protein phosphatase

  •  
  • PTM

    post-translational modification

  •  
  • PUMA

    p53 up-regulated modulator of apoptosis

  •  
  • TAD

    transactivation domain

  •  
  • TGFβ

    transforming growth factor β

  •  
  • TIP60

    60 kDa tail-interacting protein

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Author notes

1

I dedicate this article to my early mentors: Hugh Nimmo, whose undergraduate lectures on protein phosphorylation and whose support and guidance during my Ph.D. studies formed the cornerstone of my career, and John Coggins for his stimulating lectures on protein structure and function and his infectious enthusiasm for science.