Biochimica et Biophysica Acta 1602 (2002) 47^59

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Review

Activation of the p53 tumor suppressor protein

Karen H. Vousden *
Regulation of Cell Growth Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA Received 25 September 2001; received in revised form 11 December 2001; accepted 13 December 2001

Abstract The p53 tumor suppressor gene plays an important role in preventing cancer development, by arresting or killing potential tumor cells. Mutations within the p53 gene, leading to the loss of p53 activity, are found in about half of all human cancers, while many of the tumors that retain wild type p53 carry mutations in the pathways that allow full activation of p53. In either case, the result is a defect in the ability to induce a p53 response in cells undergoing oncogenic stress. Significant advances have been made recently in our understanding of the molecular pathways through which p53 activity is regulated, bringing with them fresh possibilities for the design of cancer therapies based on reactivation of the p53 response. Published by Elsevier Science B.V.
Keywords: p53; MDM2; Tumor suppression; Apoptosis; Ubiquitination; Subcellular localization; Transcriptional activation

1. Introduction The intense interest generated by p53 (almost 5000 publications last year alone) seems remarkable for a protein that is not essential for normal growth and development, and that is present at almost undetectable levels in most cells. This fascination with p53 stems from its contribution to tumor suppression, and the fact that loss of normal p53 function occurs in almost all cancers of both mice and men [1]. A consensus has emerged that p53 responds to the types of stress signals that can cause oncogenic alterations, such as DNA damage, or conditions that arise in developing tumor cells, such as abnormal proliferation or hypoxia. Activation of p53 by these stress signals rapidly inhibits cell growth, by arresting proliferation or inducing apoptosis, thereby prevent-

ing the propagation of cells that may be undergoing malignant transformation. A number of mechanisms are now being explored to use p53 to kill tumor cells [2], including the stimulation of endogenous p53 function in tumors that retain wild type p53 but are defective in their ability to activate p53. These approaches require a detailed understanding of how p53 function is regulated in cells, and rapid progress has been made in this area recently.

2. Functions of p53 p53 is an extremely ecient inhibitor of cell growth, inducing cell cycle arrest and/or apoptotic cell death, depending on cell type and environment [3]. Regulation of p53 activity is therefore critical to allow for both normal cell division and tumor suppression ^ p53 function must be dampened suciently to allow normal growth and development, while retaining the capacity for rapid induction in

* Fax: +1-301-846-1666. E-mail address: vousden@ncifcrf.gov (K.H. Vousden).

0304-419X / 02 / $ ^ see front matter Published by Elsevier Science B.V. PII: S 0 3 0 4 - 4 1 9 X ( 0 2 ) 0 0 0 3 5 - 5

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response to stress associated with tumorigenesis. Not surprisingly, therefore, there are many mechanisms through which p53 is regulated. Although control of p53 transcription and translation has been described, it seems evident that the major mechanisms through which p53 function is controlled are regulation of p53 protein levels, control of the localization of the p53 protein and modulation of the activity of p53, particularly its ability to function as a sequence specic transcription factor. The p53 protein is subject to several post-translational modications [4] (Fig. 1) and interacts with numerous other cell proteins [5], and we are now beginning to understand how these modications and protein interactions contribute to the regulation of p53 stability, location and activity.

3. Regulation of p53 protein stability through ubiquitin-dependent degradation p53 levels are kept low in most normally prolifer-

ating cells by rapid protein degradation. One of the key components regulating p53 stability is MDM2, a protein that functions as a ubiquitin ligase for p53, mediating ubiquitination of p53 and allowing it to be recognized and degraded by the proteasome [6]. MDM2 also ubiquitinates itself and regulates its own stability, although this autoubiquitination activity can be separated from the ability to target p53 for degradation (see below). MDM2 is a transcriptional target of p53, and the importance of this feedback loop, in which p53 controls the expression of its own regulator, was most elegantly demonstrated by the generation of mice that are deleted of MDM2 [7,8]. Loss of MDM2, and the resultant failure to reign in p53 activity, induces early embryonic lethality that is entirely dependent on p53 function. Interestingly, deletion of MDMX, a protein related to MDM2, also results in an embryonic lethality that is rescued by simultaneous loss of p53 [9]. These studies identify MDMX as another important regulator of p53 activity, and although MDMX does not target p53 for degradation [10,11], it can inhibit p53s ability to

Fig. 1. p53 structure and modication. The cartoon of p53 shows the N-terminal trans-activation domain, which interacts with components of the transcriptional machinery such as CBP/p300, as well as MDM2. The central sequence specic DNA binding domain contains four of the ve evolutionarily conserved regions (shown in orange) where most of the point mutants found in cancers occur. The tetramerization domain is in the C-terminus of the protein. Also shown are the nuclear localization signals (NLS) and the nuclear export sequences (NES). Multiple post-translational modications of p53 are almost all localized to the N- and C-termini of the protein. Sites of acetylation, phosphorylation, ubiquitination and Sumoylation are indicated in the expanded view of the N- and C-terminal regions of the p53 protein. Some of the lysine residues in the C-terminus have been identied as potential targets for modications by both ubiquitination and acetylation, or ubiquitination and Sumoylation, and the balance between these modications is likely to contribute to the regulation of p53 activity.

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activate transcription of target genes [12]. The relative contribution of MDM2 and MDMX in p53 regulation remains to be determined, but amplication of both MDM2 and MDMX has been found in tumors that retain wild type p53 [13,14]. These observations suggest that enhanced expression of MDM2 or MDMX prevent the activation of p53 as a tumor suppressor, and can substitute for mutational loss of p53 during tumorigenesis. The embryonic lethality of the MDM2 and MDMX null mice provides a clear illustration of the importance of curbing p53 function in normal cells. Although phosphorylation of p53 has been most extensively linked to activation of the p53 response (see below), phosphorylation may also play a role in the negative regulation of p53 stability. The COP9 signalosome (CSN) complex has recently been shown to target phosphorylation of p53 on threonine 155, leading to enhanced proteasome-dependent degradation [15]. Since CSN is constitutively active, it is possible that CSN activity contributes to the negative regulation of p53 in unstressed cells, while inhibition of CSN function may serve to stabilize p53 in response to some stress signals. Similarly, phosphorylation in the C-terminus of p53 by protein kinase C enhances the ubiquitination and degradation of p53 in unstressed cells [16], and the dephosphorylation of these sites that is seen in response to ionizing radiation [17] may contribute to the DNA damage-induced stabilization of p53. Several oncogenes that contribute to deregulated proliferation in cancers have been shown to induce p53, an activity that plays a critical role in preventing tumor development. However, these proto-oncogenes are also important components of normal cell cycle progression, posing a conundrum in which the signals that drive proliferation also signal to arrest cell growth through activation of p53. It now appears that several mechanisms exist to dampen the p53 response to allow normal proliferation to proceed. One example of this is the recent identication of MDM2 as a target for phosphorylation by the serine/threonine kinase Akt [18,19]. Akt is activated by growth factor stimulation, and phosphorylation of MDM2 is associated with nuclear localization of MDM2 and enhanced degradation of p53 [18,19]. This activity of Akt could therefore prevent activation of p53 during a normal proliferative response,

and may also contribute to the ability of Akt to protect against p53-induced cell death [20]. However, the eects of Akt are certainly more complex than simply allowing nuclear localization of MDM2, and Akt activity has also been shown to directly inhibit p53 transcriptional activity without aecting p53 stability [21]. Regulation of MDM2 stability is another way in which p53 activity can be controlled. Several mechanisms allow for the selective stabilization of MDM2, without inhibition of the ability to target p53 for degradation, resulting in enhanced degradation of p53. For example, MDM2 can be modied either by ubiquitin or Sumo conjugation. Sumo modication has been suggested to prevent auto-ubiquitination, thereby stabilizing an MDM2 protein that retains the ability to target p53 for degradation [22]. In this model, Sumoylation occurs following the binding of Ubc9, an enzyme with similarity to the ubiquitin-conjugating enzymes or E2s, to the N-terminus of MDM2 [23]. Stress-induced reduction of Ubc9 binding correlates with reduced Sumoylation, enhanced ubiquitination and degradation of MDM2, and consequently the stabilization of p53. The dissociation of MDM2 and p53 degradation is also utilized by TSG101, an MDM2 binding protein that stabilizes MDM2 and so enhances the degradation of p53 [24]. It is possible that TSG101 competes with the E2 ubiquitin-conjugating enzyme for MDM2, although how p53 degradation is maintained under these conditions is not clear. Deletion of the tsg101 gene causes an early embryonic lethality in mice associated with p53 accumulation [25], indicating that TSG101 is another important component of the machinery that keeps p53 under control in normal cells. Apart from Akt, other components of signaling pathways associated with cell proliferation, such as Src [26] or c-Jun [27], can also function to block p53 activity. This activity of c-Jun, and other AP1 family members such as JDP-2, is mediated by direct inhibition of p53 expression [27,28] and inhibition of the binding of p53 to target promoters [29]. These may play a role in both allowing normal growth and in the reversal of the p53-induced growth arrest at the end of a damage response. Interestingly, JunD has been shown to reduce expression of ARF, a small protein that inhibits MDM2 and plays a role in in-

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ducing p53 in response to oncogene activation (see below) [30]. In epithelial cells, activation of the Ras signaling pathway can stimulate JunD activity [31,32], and it is possible that in these cells JunD reduces ARF levels. The eect of this would be to increase MDM2 activity, thereby contributing to the suppression of p53 during normal cell division. Such an activity would cooperate with the ability of Ras to enhance transcription of the MDM2 gene [33]. However, this function of Ras is counter-balanced by the ability of Ras to induce ARF, and so stabilize p53 [34]. How Ras induces ARF is not clear, and may depend on cell type. For example, in broblasts Ras activation is associated with the down-regulation of JunD [35], which could lead to the enhanced expression of ARF. The opposing abilities of the same signaling pathways to both induce and inhibit p53 function reect the delicate balance between allowing normal cell replication while maintaining a rapid emergency response to terminate abnormal proliferation.

4. Stabilization of p53 in response to stress Activation of p53 in response to stress signals is almost always accompanied by stabilization of the p53 protein, and several pathways leading to the inhibition of MDM2-mediated degradation in response to stress have been described [36]. Degradation of p53 by MDM2 depends on the interaction between the two proteins, and a small domain in the N-terminus of p53 directly contacts a deep hydrophobic cleft in the N-terminus of the MDM2 protein [37]. This interaction between MDM2 and p53 can be impaired by phosphorylation of p53 within the MDM2 binding region (Fig. 1). Phosphorylation of these residues occurs in response to stress such as DNA damage, mediated at least in part by the kinases Chk1, Chk2, ATM and ATR, that are activated by genotoxic damage [4]. DNA damage-induced phosphorylation of p53 therefore allows stabilization of p53 by reducing binding to MDM2, and an interesting feedback loop is suggested by the observation that p53 expression can lead to the down-regulation of Chk1 [38]. The importance of the ATM/Chk kinase cascade in allowing activation of p53 in response to certain types of genotoxic dam-

age is illustrated most clearly by the observation that either ATM- or Chk2-decient cells are defective in their ability to stabilize p53 in response to ionizing radiation [39,40]. However, p53 may not be the only important substrate for these kinases. MDM2 has also been shown to be phosphorylated by ATM, and although this occurs at a site distant from the p53-binding region of MDM2, the result is an impaired ability of MDM2 to target degradation of p53 [41]. In the same vein, phosphorylation of the p53binding pocket on MDM2 by DNA-PK may also inhibit interaction with p53 and so inhibit degradation [42]. Numerous other kinases have also been shown to phosphorylate p53, and potentially play a role in the regulation of p53 stability [4]. For example, phosphorylation of p53 at threonine 81 by JNK has been implicated in stabilization of p53 in response to UV irradiation [43], and the Polo-like kinase-3 contributes to the phosphorylation of p53 at serine 20 (the residue targeted by Chk2) in response to reactive oxygen species [44]. Similarly, multiple phosphorylation sites in MDM2 are likely to play a role in the regulation of MDM2 function [45,46], and recently phosphorylation of mouse MDM2 by cyclin A-CDK2 in a region outside the p53-binding domain has been shown to weaken the interaction with p53 [47]. This ability of CDKs to induce p53 may be counteracted during the normal cell cycle by a contribution of CDK activity to the maintenance of MDM2 expression [48]. Although clearly important for some responses, phosphorylation does not appear to be necessary for stabilization of p53 in all situations. It is well established that dierent stress signals give rise to dierent patterns of p53 phosphorylation, and simultaneous mutation of the N-terminal phosphorylation sites in p53 does not prevent stabilization of p53 protein in response to all forms of stress [49]. Many other mechanisms that may allow stabilization of p53 have been described [36]. These include downregulation of MDM2 expression, down-regulation of the levels of free ubiquitin [50] or competition between MDM2 and transcriptional co-activators like TAFII31 and Strap that bind to the same region of p53 [51,52]. Recently, c-Abl has been shown to be necessary to allow ecient stabilization of p53 in response to DNA damage. Although c-Abl can

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form a complex with p53, it does not inhibit the p53/ MDM2 interaction, but somehow prevents ubiquitination of p53 by MDM2 [53]. Similarly, an interaction between hypoxia-inducible factor 1K (HIF1K) and p53 can stabilize and activate p53 [54]. HIF1Kinduced activation of p53 in response to hypoxia may play an important tumor suppressive role as solid cancers grow in size, and an ability of p53 to down-regulate HIF1K through MDM2 may also contribute to the suppression of angiogenesis and further tumor progression [55]. MDM2 binding proteins are also integral to the regulation of p53, including alternatively spliced forms of MDM2 itself, that sequester full length MDM2 in the cytoplasm [56], and expression of other MDM2 binding proteins such as pRB [57] or ARF [58^61]. The role of ARF is of particular interest, since this protein plays of role in the stabilization of p53 in response to the deregulated activity of many oncogenes that contribute to abnormal proliferation [62]. Although loss of ARF does not prevent stabilization of p53 in response to all stress signals, mice lacking Arf are highly tumor susceptible, and loss of ARF can, to some extent, substitute for loss of p53 [62]. An example of this is seen in the early stages of colon carcinogenesis, where deregulation of L-catenin, a signal that induces p53 through ARF, is frequently associated with ARF silencing by promoter hypermethylation [63,64]. Interestingly, the activation of p53 by L-catenin has recently been shown to be counter-balanced by the ability of p53 to down-regulate L-catenin, through activation of Siah-1, a p53-inducible gene encoding a protein that can promote the degradation of L-catenin [65,66]. Although it is becoming clear that ARF has activities that are independent of p53 [67], it seems likely that activation of p53 by ARF plays a key role in eliminating cells that develop proliferative abnormalities, thereby protecting the organism from cancer development. Although MDM2 is a principal player in the regulation of p53 stability, additional mechanisms also exist to control degradation of p53. The kinase JNK has been shown to target degradation of p53 independently of MDM2 [68], while interaction of p53 with the co-repressor mSin3a has recently been shown to protect p53 from MDM2-independent proteasome-mediated degradation [69]. How modulation

of these pathways contributes to the full activation of a p53 response remains to be fully determined.

5. Regulation of p53 subcellular localization In the past few years it has become clear that in addition to modulation of protein stability, p53 function is also regulated by controlling where the protein is in the cell. Since one of the key functions of p53 is the regulation of transcription, localization of p53 to the nucleus plays an important role in the p53 response. Active transport of p53 towards the nucleus by dynein and the microtubule network has been described [70], and several nuclear localization signals in the C-terminus of p53 can contribute to nuclear import (Fig. 1). Having entered the nucleus, regulatory mechanisms exist to control the export of p53 back out to the cytoplasm. The p53 protein contains two nuclear export sequences, one in the Cterminal oligomerization domain [71], and the other in the N-terminal MDM2 binding region [72]. The details of how p53 nuclear export is regulated remains somewhat controversial, and the development of a unied model is rather dicult. Overall it would appear that the ability of p53 to be exported is greatly enhanced by the action of MDM2 [73], although export is not absolutely dependent on the presence of MDM2 [71,72]. The ability of MDM2 to enhance p53 export depends on its function as a ubiquitin ligase [74,75], and a p53 mutant lacking the six C-terminal lysine residues that are normally ubiquitinated by MDM2 (Fig. 1) is resistant to both MDM2-mediated nuclear export and degradation [76^78]. The C-terminal nuclear export sequence in p53 is normally not accessible to the export pathway when p53 is tetramerized [71], and it is tempting to speculate that ubiquitination within this region of p53 unmasks the nuclear export sequence and allows p53 to move to the cytoplasm. ZBP-89, a protein that stabilizes p53 and inhibits nuclear export, functions by binding the central and C-terminal domains of p53, and may block ubiquitination and subsequent unmasking of the C-terminal nuclear export sequence in p53 [79]. By contrast, the activity of the N-terminal nuclear export sequence is regulated by phosphorylation within this region p53 (which is within the MDM2 binding domain) [72], and stress-

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Fig. 2. A model for the activation of p53 involving N-terminal phosphorylation. In unstressed cells, MDM2 binds to the N-terminus of p53 and ubiquitinates lysine residues in the C-terminus, targeting p53 for degradation and allowing ecient nuclear export through the C-terminal nuclear export sequence. Stress-induced phosphorylation within the N-terminus of p53 leads to several activating events. N-terminal phosphorylation of p53 can inhibit MDM2 binding, which prevents ubiquitination and so both stabilizes p53 and restricts nuclear export. N-terminal phosphorylation also inhibits the activity of the N-terminal nuclear export sequence in p53, contributing to the ecient accumulation of nuclear p53. N-terminal phosphorylation also enhances interactions of p53 with transcriptional co-activators like p300/CBP, which can acetylate residues in the C-terminus and promote p53s transcriptional activity.

induced phosphorylation inhibits nuclear export through this N-terminal signal. These observations suggest that N-terminal phosphorylation of p53 may augment nuclear retention of p53 in two ways, rstly by directly inhibiting the N-terminal nuclear export sequence, and secondly by inhibiting MDM2 binding and so reducing ubiquitination and activation of the C-terminal nuclear export sequence (Fig. 2). One slightly perplexing question is why p53 should be exported from the nucleus [80], and one suggestion has been that nuclear export is required for the degradation of p53 [81]. The simple model in which MDM2 ubiquitinates p53 and so targets p53 for recognition by the proteasome has been complicated by

the observation that MDM2 may only mono-ubiquitinate p53 [82]. This means that another enzyme in the ubiquitin system, such as another E3 or an E4, will be required to achieve poly-ubiquitination of p53 and recognition by the proteasome. The existence of a missing factor has also been suggested by the observation that some MDM2 mutants retain ubiquitin ligase activity but do not target p53 for degradation [41,83^85]. One possible candidate for this missing link is p300 [84], which was identied as playing a role in MDM2-mediated degradation of p53 that is quite distinct from its role as a transcriptional cofactor [86]. It has been suggested that p300 mediates the formation of a complex containing p53 and the 26S proteasome [84], although factors other than p300

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may also provide the link between p53/MDM2 complexes and the proteasome [85]. The observation that nuclear export contributes to p53 degradation [74,75,81] allows a further renement of this model, in which the missing factor necessary to allow degradation of p53 after ubiquitination by MDM2 resides exclusively in the cytoplasm. However, there is as yet no rm evidence to support this idea, which has been challenged by recent reports showing that export of p53 to the cytoplasm may not be necessary for either ubiquitination or degradation [78,87^89]. This leaves us with another dilemma: if nuclear export is not required for p53 degradation, why evoke such complicated mechanisms to allow p53 to leave the nucleus? One plausible suggestion is that the export of p53 represents yet another mechanism to ensure ecient control of p53 function, another safety device to prevent p53-mediated inhibition of cell growth during development. But maybe more intriguing is the possibility that nuclear export is actually required for some functions of p53. Although a somewhat heretical suggestion, at least some p53 has been shown to localize to mitochondria [90], and it is possible that p53 export to the cytoplasm can contribute to certain aspects of the apoptotic response. Nevertheless, a role for p53 in the cytoplasm is clearly secondary to its nuclear function, and nuclear localization is essential for p53 activity. Indeed failure of wild type p53 to localize to the nucleus, due to either defects in the ability to enter the nucleus or following hyperactive nuclear export, appears to contribute to the inactivation of p53 in a number of tumors [71,91,92]. In addition to nucleo-cytoplasmic shuttling, movement of p53 within the nucleus is also regulated. Under various conditions p53 has been shown to reside in several nuclear structures, including the nucleoli [93] and nuclear bodies (NBs) [94,95]. Of particular interest is the association of p53 with the PML protein, which results in the colocalization of p53 with PML and the histone acetyl transferase CBP to the PML NBs [96^99]. The interaction with PML and recruitment to the NBs enhances p53s transcriptional activity, and PML is necessary for p53 to induce apoptosis and senescence. The NBs play an important role in transcriptional regulation, and it seems likely that the interaction of p53 with other transcription factors recruited to the NBs con-

tributes to both the activation and modulation of p53s transcriptional function [100].

6. Regulation of p53 activity Regulation of p53 through protein stability and localization is potentially augmented by modulation of the ability of the p53 protein to bind DNA and interact with other components of the transcriptional machinery, activities that are also aected by posttranslational modications of p53. A well-established model based on in vitro studies suggests that p53 can exist in two conformations that are latent or active for DNA binding [101]. Proteins such as Ref-1 [102] and HMG-1 [103] can activate latent forms of p53, and these proteins contribute to the ability to induce the p53 response in cells. The C-terminal region of the p53 protein has been described as playing a key role in regulating p53s DNA binding activity, and was originally suggested to function through an allosteric mechanism, by binding to the central core domain in p53 and inhibiting sequence specic DNA binding [101]. Release of the inhibitory eect of the C-terminus was proposed to lead to a conformational shift, to allow DNA binding by the core domain of p53. More recently, alternative models for the role of the C-terminal region of p53 have been suggested. These include the binding of this region directly to DNA, thereby inhibiting, or interfering with the DNA binding by the p53 core [104,105], or interaction of the C-terminal region with another (unidentied) factor that inhibits p53s transcriptional activity [106]. This issue has been at least partially resolved by recent structural analysis of full length or C-terminally truncated p53 proteins, that are latent or active for DNA binding in the in vitro assays [107]. These studies showed no evidence for changes in the conformation when comparing the two states, questioning the original allosteric model for the regulation of p53 activity. Whatever the function of the C-terminus of p53, there are numerous covalent and noncovalent modications within this region that could modulate DNA binding activity [108]. Particularly interesting are modications such as phosphorylation, Sumoylation or even glycosylation. For example, a DNA damage-induced complex containing casein kinase 2 and the chromatin transcrip-

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tional elongation factor is responsible for UV-induced phosphorylation of serine 392 in the C-terminus of p53 [109], leading to activation of DNA binding [110]. Similarly, Sumo modication of p53 at lysine 386 within the C-terminus may also activate p53 [111^113], although this modication is not essential for p53 function [114]. Stress-induced C-terminal acetylation by acetyltransferases such as CBP/ p300 or PCAF can also activate p53 DNA binding [115^117], and inhibition of acetylation correlates with inhibition of p53 function [118^120]. However, we should bear in mind that the requirement of acetylation within the C-terminus of p53 to transcriptional activation remains controversial [96,99]. Indeed, a recent publication suggests that although binding of p53 to p300 is important for transcriptional activation, acetylation of p53 is not required for binding of p53 to its DNA binding site in chromatin, at least for the p21CIP1 promoter [121]. Rather, the role of p300 is in the subsequent acetylation of p53-bound nucleosomes, thus facilitating access by other components of the transcriptional machinery. Furthermore, the same study has even cast some doubt on whether the C-terminus of p53 really acts as a negative regulator of p53 DNA binding activity. Taken together, it seems that we can only conclude that at the moment there are no rm conclusions to be made about how, or even if, p53 activity is regulated by the C-terminal region. Moving to the other end of p53, modications in the N-terminus have also been shown to aect p53 activity. The N-terminus of p53 plays a role in regulating the p53/DNA interaction, which can be stabilized by modications within this region of the protein [122]. Phosphorylation within the N-terminus of p53 can stimulate p53s transcriptional activity without inuencing MDM2 binding, and both serine 15 and serine 33 phosphorylation leads to enhanced binding between p53 and histone acetyl transferases such as p300/CBP or pCAF [123^125]. The interactions between p53 and CBP/p300 have been associated with subsequent acetylation at the C-terminus of p53 [117,125], leading to a fairly straightforward model in which stress-induced phosphorylation at the N-terminus of p53 augments interaction with the acetyl-transferases that modify the C-terminus of p53 and activate DNA binding (Fig. 2). Nevertheless, phosphorylation of serine 15 in p53 is not necessary

for acetylation [126,127], and the p33ING2 protein, which is induced by DNA damage through p53-independent mechanisms, enhances p53 acetylation and activity without phosphorylation of serine 15 [128]. Remember, also, that acetylation of p53-bound nucleosomes in p53 target promoters, rather than acetylation of p53 itself, may be the critical event. Interestingly, binding of MDM2 directly prevents acetylation of p53, and the MDM2 binding protein ARF, which inhibits MDM2s E3 ligase activity and so leads to the stabilization of p53, also blocks this ability of MDM2 to inhibit acetylation [126].

7. Determining the choice of response The cellular response to p53 is dependent on many factors, including cell type, cell environment and whether the cell has sustained other oncogenic alterations. The enhanced sensitivity of transformed cells to undergo apoptosis in response to p53 activation, compared to their normal counterparts, is likely to make a substantial contribution to the chemosensitivity of tumor cells and the success of cancer therapy [129]. Although this sensitivity to apoptosis is regulated in part by components that cooperate with p53, it has recently become evident that modulation of p53 function itself is more complex than simply an on/o switch. The ability to induce cell cycle arrest and apoptosis appear to be dierent, separable functions of p53 [130,131], in part reecting transcriptional repression and transcriptionally independent functions of p53 that appear to contribute specically to the induction of cell death [3]. However, substantial evidence now supports the suggestion that p53 can dierentially drive cell cycle arrest or apoptosis depending on which target genes it chooses to activate. This concept was rst suggested by analysis of tumor-derived p53 mutants that showed selective defects in their ability to activate p53-responsive apoptotic promoters, correlating with a specic loss of the ability to induce cell death [132,133]. More recent studies have thrown some light on how this promoter choice might be made. One series of reports indicate that the choice is made by interaction of p53 with specic transcriptional cofactors. p53 has been shown to interact with numerous proteins that enhance its transcriptional ac-

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tivity, including p300/CBP [134^136], Zac1 [137], YB-1 [138], and AMF1 [139]. Of particular interest, however, are those transcriptional co-activators, like ASPP [140] and JMY [141], that target p53 to induce the activation of apoptotic genes, and so drive the induction of cell death. The activity of the ASPP proteins, ASPP1 and ASPP2, are particularly interesting, since they interact with p53 in the DNA binding domain and enhance binding of p53 specically to apoptotic targets. ASPP expression is required for p53-induced apoptosis, and the expression of ASPP was frequently found to be down-regulated in breast cancers that retain wild type p53, revealing another mechanism by which tumors can avoid p53-induced death. Modulation of the availability of these cofactors, or regulation of their ability to interact with p53 could be the key to determining whether a cell undergoes apoptosis or cell cycle arrest in response to p53inducing stress (Fig. 3). Interestingly, localization of p53 to NBs by PML can also regulate promoter choice by p53 [96,98], potentially through allowing interaction with cofactors that are also recruited to these locations in the nucleus. These results raise the possibility that modication of p53 could regulate interaction with these critical cofactors, and much interest has focused recently on the phosphorylation of p53 on serine 46, a region outside the MDM2 binding domain and not obviously involved in the regulation of p53 stability. Although the kinase responsible has not been denitively identied, phosphorylation at serine 46 is required for p53 to induce expression of apoptotic genes like p53AIP1 [142]. Interestingly, regulation of serine 46 phosphorylation is carried out by proteins that are themselves the products of p53-inducible genes, such as p53DINP1, which is required to activate phosphorylation at serine 46 [143], and Wip1, which functions as a phosphatase to inhibit p53 apoptotic function by dephosphorylating serine 46 [144] (Fig. 3). Phosphorylation within the C-terminus of p53 may also dierentially regulate DNA binding [145], and it is likely that several modications of p53 will contribute to the control of which genes are transcriptionally activated by p53. The regulation of which response is induced by p53 may also be inuenced by control of activities of p53 that do not depend on activation of transcription. For example, interaction of p53 with the Peutz^

Fig. 3. Regulation of the choice of response to p53. Activation of p53 leads to the induction of genes that mediate cell cycle arrest. However, additional signals that can lead to the phosphorylation of p53, the availability of certain transcriptional cofactors such as ASPP or JMY, and localization of p53 to NBs can modify the response to allow activation of apoptotic target genes and induction of programmed cell death. These responses are controlled to some extent by feedback loops, since both phosphorylation and dephosphorylation can be regulated by proteins (p53DINP1 and WIP1) that are products of p53-inducible genes.

Jegher gene product LKB1, a protein kinase with tumor suppressor activity, appears to be necessary for LKB1 translocation to the mitochondria and induction of cell death [146]. How p53 carries out this function, or how the interaction is regulated is not known, but these observations provide yet another layer to the complexity of the p53 response.

8. Conclusion p53 is a key component of the pathways regulating cellular responses to stress, and increasingly complex networks of mechanisms that contribute to the positive and negative control of p53 function are being described. As these details are uncovered, it is becoming evident that many cancers that retain wild type p53 are defective in their ability to activate the stress response, leading to a failure to properly stabilize, localize or activate p53. These defects are seen in a substantial proportion of human cancers which retain wild type p53, and therefore represent excellent candidates for treatment with drugs designed to induce p53, for example by inhibition of MDM2.

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Although activation of p53 in normal tissue will be unavoidable, there is hope that the sensitization of malignant cells to p53-induced apoptosis will allow selective killing of the tumor [129]. The clear advantage of such approaches, compared to many conventional chemotherapies available at the present, is the decreased risk of genotoxic damage and subsequent undesirable long-term eects. Whether any of the eorts currently underway in this arena will successfully translate to clinical application remains to be seen, but at the rate of research currently underway there will be no shortage of novel targets for drug discovery in the p53 eld.

Acknowledgements I am extremely grateful to Marion Lohrum, Xin Lu and Moshe Yaniv for their invaluable comments on the manuscript. References
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