p53 ubiquitination by Mdm2: A never ending tail?

DNA Repair 8 (2009) 483–490

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DNA Repair journal homepage: www.elsevier.com/locate/dnarepair

Review

p53 ubiquitination by Mdm2: A never ending tail? Amanda S. Coutts, Cassandra J. Adams, Nicholas B. La Thangue ∗ Medical Sciences Division, Department of Clinical Pharmacology, University of Oxford, Oxford OX3 7DQ, UK

a r t i c l e

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Article history: Available online 12 February 2009 Keywords: p53 Ubiquitin Mdm2 DNA damage

a b s t r a c t p53 function is of critical importance in suppressing human cancer formation, highlighted by the fact that the majority of human tumors harbor compromised p53 activity. In normal cells, p53 is held at low levels in a latent form and cellular stress results in the rapid stabilization of p53. Mdm2 mediates ubiquitindependent degradation of p53 which plays a key role in maintaining cellular p53 levels. Ubiquitination was, until recently, considered a straightforward system involved in p53 degradation, but recent work has demonstrated how ubiquitination can alter p53 activity, not stability. In this review we summarize current understanding on p53 ubiquitination by Mdm2 with a particular focus on how the balance between protein levels and other post-translational modifications will direct the p53 response. © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Mdm2 oncoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Poly-ubiquitination and degradation of p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mono-ubiquitination of p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competition with other post-translational modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Mdm2 E3 ligase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Mdm2 levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Post-translational modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Recruitment of co-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p53 de-ubiquitination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction p53 is a critical human tumor suppressor and is one of the most commonly mutated genes in human cancer, with approximately 50% of cancers containing p53 mutations, while the other half is thought to contain alterations in components of the p53 pathway [1]. Genotoxic stress, such as DNA damage, results in upregulation of p53, thus allowing p53 to carry out its activity vital for the checkpoint response and suppression of tumorigenesis. The checkpoint response to DNA damage involves a myriad of cellular activities including cell cycle arrest, DNA repair and apoptosis, and defects

∗ Corresponding author. E-mail address: [email protected] (N.B. La Thangue). 1568-7864/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2009.01.008

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in the DNA damage response pathway lead to tumor development [2]. p53 is a nuclear transcription factor that affects cellular functions which include transcription, DNA synthesis and repair, cell cycle arrest, senescence and apoptosis [3]. Under normal conditions, p53 is held in an inactive state but undergoes a significant increase in protein stability upon exposure to DNA damage. DNA damage stabilizes p53 in part via the DNA damage signaling pathway that involves the sensor kinases, including ATM and ATR, and effector kinases, like Chk1 and Chk2, and leads to the transcriptional regulation of a variety of genes involved in cell cycle control and apoptosis [2,4]. The regulation of p53 is tightly controlled at numerous levels and, owing to the critical nature of p53 as a human tumor suppressor, the regulation of p53 is one of the most important systems in tumorigenesis. Whilst an increasing array of co-factors is

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Fig. 1. Schematic models depict Mdm2 and p53 structures. (A) Mdm2 domain structure and interacting proteins are indicated. The model highlights some of the interacting proteins and is not meant to be exhaustive. The N-terminal p53 binding site is indicated with gold shading. The central acidic domain comprises amino acids 237–288. The zinc finger and RING finger domains are indicated with blue and gray shading, respectively. NLS: nuclear localization signal, NES: nuclear export signal, NoLS: nucleolar localization signal. (B) p53 is comprised of an N-terminal transcriptional activation domain (TAD), a proline rich domain (PRD), a central DNA-binding domain (DBD), and C-terminal tetramerization (TD) and regulatory (CRD) domains. Sites of post-translational modifications are indicated and are not meant to be exhaustive. Putative ubiquitination sites in the DBD are marked with a question mark. Regions of some p53-interacting proteins are demonstrated. S: serine, T: threonine, K: lysine, A: arginine, Ub: ubiquitination, Ac: acetylation, Me: methylation, P: phosphorylation, NPM: nucleophosmin.

known to influence p53 activity, post-translational modification also plays an important role in p53 regulation. p53 is subject to a variety of post-translational modifications, including phosphorylation, sumoylation, acetylation, methylation and ubiquitination, which all impact significantly on p53 activity [5]. Ubiquitination of p53 has emerged as a fundamental mechanism of p53 control. p53 can be modified by several E3 ubiquitin ligases, including Pirh2, COP1, ARF binding protein and E6AP [6,7]. The oncoprotein murine double minute 2 (Mdm2 or Hdm2 in humans; hereafter referred to as Mdm2) is a key negative regulator of p53 activity and the most extensively studied of the p53 E3 ligases. The regulation of p53 ubiquitination by Mdm2 is the subject of this review. 2. The Mdm2 oncoprotein The importance of Mdm2 in regulating p53 activity is underscored by the finding that mdm2−/− mouse embryos die early in development due to massive p53-dependent apoptosis, which is rescued in the mdm2−/− /p53−/− knockout mouse [8–10]. Furthermore, because mdm2 transcription is regulated by p53, transcriptional activation by p53 provides an autoregulatory feedback loop [11]. Mdm2 is a member of the RING finger family of E3 ligases (Fig. 1A; [12,13]). The RING finger binds to E2 ubiquitin-conjugating enzymes to promote and direct ubiquitination of target proteins [14]. Ubiquitin is often conjugated to proteins in the form of a polymer, in which additional ubiquitin moieties are ligated, via one of seven lysine residues in the previously attached ubiquitin, to an existing ubiquitin molecule (referred to as poly-ubiquitination). Poly-ubiquitination serves as a signal for proteasomal degradation, with a chain four ubiquitin moieties being the minimum

required [15]. This is in contrast to the conjugation of a single ubiquitin molecule to one or more lysine residues within a target protein (referred to as mono-ubiquitination). Mono-ubiquitination has been shown to be involved in a variety of cellular processes, including protein trafficking, DNA repair and transcriptional regulation [16]. Mdm2 binds to p53 and is thought to repress p53 activity via two main mechanisms: by promoting degradation and blocking p53 transcriptional activation [12,13,17]. Recent research has shown that binding of Mdm2 to p53 without E3 ligase activity was not enough to inhibit p53 activity [18], suggesting that the E3 ligase function of Mdm2 is of primary importance in the negative regulation of p53. The N-terminal region of Mdm2 contains the most well-defined p53 interaction site [19,20] as well, the central acidic domain of Mdm2 provides a second p53 binding site (Fig. 1A; [21,22]). While initial studies on the p53-Mdm2 interaction identified the N-terminal region in p53 as a binding site for Mdm2 [23,24], subsequent studies have identified a second binding site for Mdm2 within the DNA-binding domain of p53 (Fig. 1B; [21,25]). Interestingly, mutant forms of p53 that contain single point mutations within this region have increased levels of Mdm2-dependent ubiquitination, but not degradation [25]. More recent studies have proposed a mechanism in which the binding of Mdm2 to the N-terminus is essential, but not sufficient, to signal p53 ubiquitination. This interaction causes a conformational change in Mdm2 that enables binding of the central acidic region in Mdm2 to a ‘ubiquitination signal’ in the core DNA-binding domain of p53 [26]. It is now becoming increasingly clear that the ubiquitination of p53 by Mdm2 is more complex than initially thought and serves not only to degrade p53, but also influences p53 localization and activ-

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ity, dependent on the type of ubiquitination (poly versus mono) and the levels of Mdm2. 3. Poly-ubiquitination and degradation of p53 In non-stressed cells, p53 is maintained at low levels, due to rapid degradation of p53 via poly-ubiquitination, primarily mediated by high basal levels of Mdm2. Early studies demonstrated that Mdm2 contained, within its RING finger domain, E3 ligase activity sufficient for poly-ubiquitination of p53, resulting in its degradation [27–29]. The precise location of p53 degradation has been the subject of much debate. It was thought that Mdm2-mediated nuclear export of p53 and subsequent cytoplasmic degradation by the 26S proteasome [30,31]. Indeed, an intact Mdm2 RING finger domain was shown to be required for efficient nuclear export of p53, suggesting that ubiquitination and nuclear export of p53 are closely linked [32]. In addition, inhibition of nuclear export with leptomycin B treatment resulted in nuclear accumulation of p53 [33]. This led to the assumption that poly-ubiquitinated p53 was exported and degraded in the cytoplasm. However, it has also been shown that nuclear export is not required for p53 degradation and that Mdm2 can promote p53 ubiquitination and degradation either in the nucleus or the cytoplasm [34,35]. It is likely that while ubiquitination and nuclear export of p53 are separable processes, mono-ubiquitination aids in the efficient export of p53 (Fig. 2; [36]; see later section). Thus, the ability of a cell to degrade p53 in either the cytoplasm or nucleus adds another layer of complexity to p53 control (Fig. 2). For example, Shirangi et al. have shown that nuclear degradation of p53 occurs during the down-regulation of the p53 response, which provides a mechanism for faster control when high p53 levels might be deleterious [37]. It is thought that Mdm2-mediated proteasomal degradation of p53 plays a critical role in suppressing p53 activity during these latter stages of the DNA damage response when high levels of Mdm2 (through p53 activation of the mdm2 gene) result in p53 poly-ubiquitination [37]. While it was clear that Mdm2 played an important role in the ubiquitination of p53, it was less clear what role Mdm2 played in the in vivo mono-ubiquitination versus poly-ubiquitination of p53. Some studies suggested that Mdm2 was not able to polyubiquitinate p53 in vitro [38]. In contrast, other studies had shown that Mdm2 alone was capable of in vitro poly-ubiquitination of p53 (see for example, [36,39]). Li et al. were able reconcile these differences when they demonstrated that Mdm2 was in fact able to both mono- and poly-ubiquitinate p53; at lower Mdm2

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levels p53 was only mono-ubiquitinated, while at higher levels poly-ubiquitination of p53 was observed, both in vitro and in vivo [40]. In addition, Mdm2-mediated poly-ubiquitination of p53 is enhanced by the recruitment of co-factors, such as p300 (see later section), adding a further layer of complexity to p53 regulation by Mdm2. 4. Mono-ubiquitination of p53 In contrast to poly-ubiquitinated p53, which is unstable and degraded by the proteasome, studies have demonstrated that mono-ubiquitinated p53 is stable (e.g., [41]), suggesting that monoubiquitinated p53 has a distinct role in p53 function. In unstressed cells p53 shuttles constantly between the cytoplasm and the nucleus, mediated by its nuclear export (NES) and import (NLS) signals (Fig. 1B). As discussed, lower levels of Mdm2 promote monoubiquitination of p53, and this has been shown to be sufficient for nuclear export of p53 [40]. In vitro studies demonstrated that Mdm2 can mono-ubiquitinate six lysine residues (370, 372, 373, 381, 382, 386) in the C-terminal region of p53 (Fig. 1B; [38,39]). Ubiquitination of the C-terminal region in p53 is thought to expose the C-terminal NES, leading to p53 nuclear export [36,40,42]. p53 also contains a cluster of five lysines within its DNA-binding domain, and some evidence suggests that Mdm2 can also ubiquitinate p53 within the DNA-binding domain, although the precise lysine residues involved have not yet been identified [43,44]. Ubiquitination of p53 at both the C-terminal region and the DNA-binding domain has been shown to contribute to its nuclear export [44]. Furthermore, Carter et al. provided data in support of a model in which p53 nuclear export is a two-step process requiring first monoubiquitination by Mdm2, resulting in exposure of the NES, followed by a subsequent additional modification of p53 (e.g., sumoylation), allowing release of Mdm2 [45]. A population of stable mono-ubiquitinated p53 molecules might therefore contribute to a cytoplasmic p53 function (Fig. 2). While p53 has a well-defined role in cell cycle arrest or apoptosis via its nuclear activity as a transcription factor, it is also apparent that p53 plays a role in apoptosis through transcriptionindependent mechanisms. Numerous studies have demonstrated a role for p53 at mitochondria, where it accumulates during stress to control mitochondrial-directed apoptosis (e.g., [46–49]). How p53 was directed to the mitochondria was unclear, but recent research has demonstrated that Mdm2-mediated monoubiquitination of p53 promotes translocation of p53 to the mitochondria [41]. Moreover, p53 export from the nucleus

Fig. 2. Multiple roles of p53 ubiquitination. The model depicts potential outcomes of p53 ubiquitination. p53 can be poly-ubiquitinated and targeted for proteasomal degradation either in the nucleus or the cytoplasm. Mono-ubiquitination of p53 may aid in nuclear export of p53. Research also suggests that a stable pool of monoubiquitinated p53 exists that is involved in mitochondrial p53 function. In addition, research suggests that p53 ubiquitination may be involved in p53’s role as a nuclear transcription factor.

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contributed little to the mitochondrially localized p53, supporting the existence of a pre-existing cytoplasmic pool of p53 [41].

5. Competition with other post-translational modifications p53 is subject to a plethora of post-translational modifications, including acetylation, methylation and phosphorylation. Lysine residues can be modified not only by ubiquitination, but also other post-translational modifications, including methylation and acetylation [50]. p53 is acetylated by p300/CBP in response to cellular stress, and this stress-induced acetylation is reversible and transient [51]. Of note, acetylation of p53 occurs at several lysine residues clustered in the C-terminal region that overlap with those residues targeted by ubiquitination (Fig. 1B; [52,53]), and acetylation is thought to control p53 stability by interfering with Mdm2-mediated ubiquitination [54,55]. Additionally, acetylation of p53 can decrease the p53-Mdm2 interaction [56]. It has been shown that Mdm2 suppresses p53 acetylation in part by inhibiting p300/CBP acetyltransferases [51,57]. In addition, Mdm2 recruits the histone deacetylase HDAC1 to p53, resulting in p53 deactylation [54]. Together, the evidence supports the hypothesis that acetylation promotes stabilization by blocking ubiquitination and that the coupling of deactylation and ubiquitination results in Mdm2 being able to degrade and downregulate p53 activity [54]. The importance of acetylation in p53 function is underscored by the finding that it is indispensable for p53 activation and both growth arrest and apoptosis induced by p53 [56]. More recent work has demonstrated that p53 can also be acetylated at several lysine residues within the DNA-binding domain (Fig. 1B; [56,58,59]), and it will be interesting to see whether acetylation at these sites also competes with ubiquitination. Intriguingly, p53 has recently been shown to be methylated (Fig. 1B; [60,61]). Set7/9 was able to methylate p53 at lysine 372, resulting in increased nuclear p53 stability and activation [60]. As this residue is also targeted for ubiquitination (Fig. 1B), this implies an interplay between p53 methylation and ubiquitination, although this has not as yet been thoroughly explored. Ivanov et al. have examined p53 ubiquitination in relation to lysine 372 and 373 to demonstrate that mutating these residues did not alter in vivo p53 ubiquitination levels [62], although this does not necessarily address a direct role for methylation in influencing p53 ubiquitination. Interestingly, an interplay between p53 methylation and acetylation has been demonstrated. Methylation of p53 by Set7/9 is damage-responsive and appears to precede acetylation and stabilization of p53 [62]. Methylation of p53 by Set7/9 has also been shown to mediate p53 acetylation by the acetyltransferase Tip60, which appears to bind preferentially to p53 methylated at lysine 372 [63]. Moreover, p53 is methylated at lysine 370 by Smyd2, which appears not to alter p53 levels but acts in a repressive manner to decrease p53 DNA-binding ability [61]. The methyltransferase PRMT5 methylates p53 at three arginine residues (333, 335, 357) within the oligomerization domain to influence the outcome of the p53 response [64]. As methylation at these sites influences p53 target gene specificity and stability, it will be interesting to see whether arginine methylation can impact on p53 ubiquitination and mitochondrial function. p53 is phosphorylated on more than 20 residues, and stressinduced phosphorylation is thought to play an important role in p53 stabilization and activation (reviewed in [65]). Mdm2 binds to the N-terminal region of p53, and phosphorylation of serine 15 and serine 20 within the N-terminus of p53 during cellular stress can block Mdm2 binding, although conflicting results have been reported (for example, [65,66]). Phosphorylation of threonine 18 in p53 can block Mdm2 binding to p53 [24,66,67]. Stress-induced

p53 phosphorylation of the N-terminal region is thus, in general, thought to reduce Mdm2 binding and promote binding of co-factors such as p300 [65]. Phosphorylation of p53 thus influences p53 acetylation. For example, acetylation of p53 at lysine 320 and 382 has been shown to require phosphorylation of p53 at serine 15 in response to ionizing radiation [68]. Phosphorylation of p53 therefore influences p53 ubiquitination by decreasing the Mdm2-p53 interaction as well as favoring acetylation by co-factors such as p300. Moreover, recently it has been shown that a region within the p53 DNA-binding domain that binds to Mdm2 to mediate ubiquitination is also a docking site for the calcium calmodulin regulated kinases, CHK1, CHK2 and DAPK-1, that phosphorylate serine 20 of p53, suggesting that competition for binding partners will also influence ubiquitination versus phosphorylation of p53 [69]. 6. Regulation of Mdm2 E3 ligase activity 6.1. Mdm2 levels As it is the cellular level of Mdm2 that will lead to either monoor poly-ubiquitination of p53, Mdm2 levels will impact significantly on the outcome of the p53 response. Importantly, levels of Mdm2 are regulated by p53 where it drives the expression of the mdm2 gene through a negative feedback loop [70]. A subset of human tumors has been shown to contain amplifications of the mdm2 gene, leading to over-expression of Mdm2 [71–74]. Also, a naturally occurring polymorphism within the mdm2 promoter leads to increased Mdm2 protein in human populations [75]. High Mdm2 levels would thus attenuate the p53 response to stress and may account for variations in some individuals’ susceptibility to cancer and accelerated tumor formation in humans [75]. 6.2. Post-translational modifications Like many other proteins, Mdm2 is subject to a variety of posttranslational modifications; the influence of many of these on Mdm2 function are not yet fully understood (reviewed in [76]). Mdm2 is phosphorylated at numerous sites in vivo, and phosphorylation of Mdm2 has been shown to impact on its ability to influence p53 activity [76]. For example, phosphorylation of serine 395 by ATM inhibits its ability to mediate p53 degradation and nuclear export [77,78] and phosphorylation of tyrosine 394 by c-Abl is thought to block the ability of Mdm2 to down-regulate p53 levels [79,80]. In contrast, phosphorylation at other sites on Mdm2, such as serine 166 and serine 186, has been shown to positively influence the ability of Mdm2 to poly-ubiquitinate p53 and promote degradation [81,82]. Mutation of several serine residues within the central acidic region of Mdm2 resulted in decreased ability to degrade, but not ubiquitinate p53, consistent with the idea that hypo-phosphorylation of the Mdm2 acidic region during DNA damage can decrease Mdm2 activity, resulting in increased p53 levels [83]. Phosphorylation of the central acidic domain within Mdm2 has been shown to increase p53 binding to Mdm2, and this region is also required for Mdm2-mediated p53 poly-ubiquitination and degradation [22]. Mdm2 can also act as an E3 ligase on itself, resulting in autoubiquitination and proteasomal degradation [28,29], and proteins that interact with Mdm2 have been shown to influence its autoubiquitination activity. For example, the de-ubiquitinating enzyme HAUSP (herpes virus-associated ubiquitin-specific protease) in conjunction with DAXX (death-domain-associated protein) has been demonstrated to inhibit Mdm2 auto-ubiquitination and promote Mdm2-mediated p53 degradation [84]. In addition, the

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tumor suppressor RASSF1A, by disrupting the interaction between Mdm2, DAXX and HAUSP, enhances Mdm2 auto-ubiquitination, thus favoring p53 stabilization during the DNA damage response [85]. SUMO modification of Mdm2 has been shown to play a role in modulating Mdm2 E3 ligase activity. Mdm2 is sumoylated, and sumoylation of Mdm2 decreased Mdm2 self-ubiquitination activity [86,87]. Moreover, Mdm2 sumoylation is decreased during the DNA damage response, suggesting that sumoylation may hinder ubiquitination of Mdm2, leading to increased Mdm2 levels [87]. Mdm2 has also been shown to be ubiquitinated by the acetyltransferase PCAF (p300-CBP-associated factor) [88]. Linares et al. demonstrated that PCAF stimulated Mdm2 ubiquitination in vitro and in vivo and that, surprisingly, PCAF appears to itself contain intrinsic ubiquitination activity [88]. The ability of PCAF to ubiquitinate Mdm2 was not due to stimulation of Mdm2 autoubiquitination activity and appears to be necessary for Mdm2 degradation during cellular stress and therefore subsequent p53 stability and activity [88]. 6.3. Recruitment of co-factors The acetyltransferases p300/CBP are ubiquitous transcriptional co-activators that interact with a wide variety of transcription factors, including p53. p300/CBP binds to p53 to stimulate transcriptional activity [52,89–92]. The C-terminal region of p53 contains two sites for acetylation by p300/CBP [53], and after DNA damage the association between p300/CBP and p53 increases, resulting in increased p53 acetylation [93]. In addition, p300 binds to Mdm2, and a role for p300 in the Mdm2-dependent regulation of p53 levels has been suggested [94,95]. Of note, Mdm2-derived mutant proteins defective in p300, but not p53, binding were unable to promote p53 degradation [94]. Zhu et al. subsequently demonstrated that a mutant Mdm2, defective in p300 binding, was able to promote p53 ubiquitination, but not degradation [96], demonstrating that ubiquitination and degradation are independent processes. Interestingly, while p300 alone displayed no effects on p53 ubiquitination in vitro, in the presence of Mdm2 it resulted in poly-ubiquitinated p53 species under conditions where Mdm2 resulted in only monoubiquitinated p53, suggesting that the intrinsic ubiquitin ligase activity of p300 is required for p53 poly-ubiquitination and turnover [97]. Moreover, the RING finger domain of Mdm2 can be acetylated by p300/CBP, which is thought to compromise Mdm2 E3 ligase activity, leading to decreased p53 poly-ubiquitination and increased p53 levels [98]. As already discussed, acetylation of p53 by p300 may also prevent ubiquitination and lead to p53 stabilization. Thus, p300 may play dual roles in p53 regulation, either increasing or decreasing poly-ubiquitination perhaps dependent on the cellular background and the nature of the stress response. YY1 (Yin Yang 1), a multifunctional transcription factor, can enhance Mdm2-mediated p53 poly-ubiquitination both in vivo and in vitro [55,99]. YY1 increases the physical association between Mdm2 and p53, ultimately leading to decreased p53 levels and activity [99]. YY1 can also bind to p53 and inhibit its transcriptional activity by preventing the recruitment of p300, which also results in prevention of p300-mediated p53 acetylation and stabilization [100]. A variety of other proteins have been shown to affect the p53/Mdm2 pathway during the cellular response to stress. For example, the tumor suppressor ARF (p14ARF in humans and p19ARF in mouse), can increase p53 levels and activity, in part by inhibiting Mdm2 activity and sequestering Mdm2 in the nucleolus [101–106]. Other studies have shown that ARF can inhibit Mdm2 auto-

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ubiquitination activity in vitro [105,107]. The interaction of ARF with Mdm2 results in decreased p53 poly-ubiquitination and increased Mdm2 levels, but this is not associated with a decrease in Mdm2 ubiquitination in vivo [104]. ARF has also been demonstrated to enhance the sumoylation of Mdm2, but this did not appear to alter Mdm2 E3 ligase activity [108], and ARF and Mdm2 co-expression can also enhance p53 sumoylation [109]. ARF-mediated sumoylation of Mdm2 and p53 may therefore be related to the ability of ARF to relocalize Mdm2 to the nucleolus. Other nucleolar proteins have been shown to influence p53 stability. For example, the nucleolar protein nucleophosmin exits the nucleolus during DNA damage and binds to both p53 and Mdm2, leading to p53 stabilization and activation [110,111]. Moreover, competition is certain to exist between different interacting proteins that will influence the outcome of the p53 response. For example, ARF has been shown to interact with YY1, disrupting the interaction between YY1 and Mdm2 [99]. The nuclear co-repressor KAP1 has been described as an Mdm2interacting protein [112]. KAP1 interacts with Mdm2 via the central acidic domain and stimulates formation of the p53-HDAC1 complex, leading to p53 ubiquitination and degradation [112]. Interestingly, KAP1 was shown to reduce YY1-Mdm2 binding, and ARF competes with KAP1 for Mdm2 binding [112]. The central acidic domain of Mdm2 interacts with multiple proteins (Fig. 1A), and as such the cellular background is likely to contribute significantly to the ability of Mdm2 to regulate p53. Mdmx (also called Mdm4) is an Mdm2 family member that also contains a RING finger domain. The exact function of Mdmx is poorly understood, and despite its RING finger domain and the ability to function as an ubiquitin ligase in vitro [113], it does not ubiquitinate and degrade p53 in cells [114,115]. Mdmx−/− mice exhibit a lethal phenotype that is rescued by inactivation of p53, stressing the fact that Mdmx, like Mdm2, is also a critical regulator of p53 [116,117]. Mdmx can bind directly to p53 and inhibit p53 transcriptional activity, and this appears to be a primary mechanism in Mdmx inhibition of p53 [118,119]. So although there appear to be differences in the roles of Mdm2 and Mdmx in regulating p53 (i.e., inhibition of trans-activation by Mdmx and targeted degradation by Mdm2), there is evidence for the functional interplay between the two proteins. Mdm2 and Mdmx interact via their RING finger domains, and the interaction of Mdmx with Mdm2 protects Mdm2 from degradation [120,121]. In fact, Mdmx can stabilize both p53 and Mdm2 [115,121]. Studies have also shown that Mdm2 is involved in the regulation of Mdmx levels [122,123]. Mdmx has been shown to cooperate with Mdm2 to target p53 in vivo [124,125], and the Mdm2-Mdmx complex is a more efficient and abundant E3 ligase complex than Mdm2 alone [126]. Therefore, the stoichiometry of the different cellular complexes is going to dictate the outcome of either Mdm2 or Mdmx expression. In addition, the effect of other interacting proteins is likely to influence the situation. For example, like Mdm2, Mdmx can associate with ARF and block ARF-mediated p53 activation [127,128], and co-expression of Mdmx and ARF decreases Mdm2 protein levels [128]. Moreover, Mdmx is also a substrate for ARF-mediated sumoylation, and increased Mdmx over-expression leads to a loss of ARF-mediated Mdm2 sumoylation and stabilization [129] Again, the complexities involved and the cross-talk within these different protein complexes need to be more thoroughly investigated before we can fully appreciate how the regulation of the p53 response is fine-tuned. The ribosomal proteins L5, L11, and L23 have all been shown to be binding partners of Mdm2, and they function by binding to the central acidic and zinc finger region in Mdm2 to inhibit E3 ligase activity of Mdm2. This may play an important role in p53 regulation during ribosomal stress [130–135].

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7. p53 de-ubiquitination The discovery of de-ubiquitinating enzymes (DUBs) demonstrated the dynamics of the ubiquitin–proteasome system. The DUB HAUSP can de-ubiquitinate p53 both in vitro and in vivo to stabilize p53 levels [136]. Surprisingly, despite de-ubiquitinating and stablizing p53, reduction of HAUSP by RNAi or genetic ablation in cells results in stabilization and activation of p53 [137,138]. This paradoxical observation appears to be due to the fact that HAUSP also targets Mdm2 and has been shown to interact with Mdm2 to de-ubiquitinate and stabilize it [137,138]. This demonstrates yet again the complexity in the regulation of p53 by the ubiquitin–proteasome system and suggests that levels of HAUSP will influence the balance between p53 activation by de-ubiquitination or degradation through enhanced Mdm2 activity. Indeed, it appears that partial reduction of HAUSP in some cell types results in p53 destabilization, while a full reduction results in Mdm2 destabilization and enhanced p53 levels [138]. HAUSP has also been shown to play a role in p53’s mitochondrial activity. Mitochondrially localized HAUSP forms a stress-induced complex with p53, resulting in p53 de-ubiquitination, which is thought to result in apoptotically active mitochondrial p53 [41].

8. Conclusions p53 ubiquitination was at one time considered to be a fairly straightforward process with the outcome being proteasomal degradation. Perhaps not surprisingly for such a critical cellular protein, the regulation of p53 by ubiquitination seems all but straightforward. As recent research demonstrates we now know that mono-ubiquitinated p53 is stable and monoubiquitination plays a role in targeting p53 to the mitochondria [41]. In addition, several other E3 ligases that can target p53 for ubiquitination have been identified; the relative importance and significance of these remain to be fully clarified. Of particular interest is the recent work by Le Cam et al. demonstrating that p53 ubiquitination by the transcription factor E4F1 recruits p53 to chromatin and promotes transcriptional activation [139], further demonstrating that ubiquitination is involved in much more than targeting p53 for destruction (Fig. 2). Many questions remain regarding the control of p53 by ubiquitination and the role that Mdm2 plays in this. For example, isoforms of p53 exist (reviewed in [140]), but how these isoforms are modulated and impact on the p53 response remains to be fully elucidated. There is evidence that some p53 isoforms can impact on p53 function (see for example, [141]). Moreover, numerous Mdm2 splice variants exist [142]. p53 is mutated in nearly 50% of human cancers, and some studies have shown differential degradation of mutant and wild-type p53 by Mdm2 (for example, [143]). Therefore, understanding the differences in regulation of mutant and wild-type p53 proteins is of significant importance, especially in light of the fact that many therapies are aimed at reactivating p53 in human tumors. A theme that emerges throughout is the fact that p53 activity reflects a delicate balance between different post-translational modifications, ultimately controlled by a vast array of co-factors and interacting partners, the levels of which crucially add another layer of complexity to the outcome. In this regard, the cellular background and the nature of the stress signal will significantly impact on the p53 response. The ability of a cell to respond in a fashion that will tip the scale in favor of p53 degradation versus monoubiquitination leading to other cellular functions, such as apoptosis, could be a matter of cellular life or death.

Conflict of Interest None.

Acknowledgements We apologize to those whose work could not be cited due to space constraints. Work in our laboratory is supported by the MRC, CRUK, LRF, AICR and EU.

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