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Biochemical Society Transactions (2010) Volume 38, part 1

Lessons from interconnected ubiquitylation and acetylation of p53: think metastable networks Monsef Benkirane*, Claude Sardet† and Olivier Coux‡1 *IGH, CNRS, UPR 1142, 141 rue de la Cardonille, 34396 Montpellier cedex 5, France, †IGMM, CNRS, UMR 5535, Montpellier University, 1919 route de Mende, 34293 Montpellier cedex 5, France, and ‡CRBM, CNRS, UMR 5237, Montpellier University, 1919 route de Mende, 34293 Montpellier cedex 5, France

Abstract The critical tumour suppressor p53 plays a major role in response to DNA damage and, more generally, to genotoxic stress. The regulation of its expression and functions is under very tight controls, and involves, in particular, an extremely complex set of post-translational modifications, thanks to a variety of ‘modifiers’, including ubiquitylation E3s and acetyltransferases, that fine-tune the stability and activity of the protein. Work of the last few years has revealed that, in addition to targeting p53, these modifiers also modify each other, forming an intricate network of regulatory molecules and events that must be taken into account to understand p53 regulation. We propose that this network allows a metastable equilibrium that confers both sensitivity and robustness on the p53 pathway, two properties that allow the pathway to respectively answer to a variety of stimuli and return to its initial stage when the stimuli disappear.

Introduction The p53 protein is a key transcription factor activated by a variety of cellular stresses that plays a central role in the protection of multicellular organisms against genome insult (hence its nickname, ‘guardian of the genome’) [1,2]. Schematically, the primary function of p53 is to stop the proliferation of cells with damaged DNA, in order to prevent propagation of their altered or abnormal genome. As tumour progression is, in most cases, associated with genome instability, p53 is thus a potent tumour suppressor: in fact, its anti-tumoral effect is so important in humans that virtually no cancer can occur without a functional defect of the protein (50% of human cancers) or of its upstream regulators (most of the other cancers) [3]. Once activated by stress, depending on the damage and the cell context, p53 inhibits proliferation by causing cell-cycle arrest, apoptosis or senescence. However, how these outcomes are controlled remains largely obscure. Nonetheless, what seems clear is that post-translational modifications of the protein are key determinants in this process. Indeed, an accepted, although simplified, model of p53 activation is that cellular stress triggers stabilization and thus accumulation of the protein, which then initiates a cytostatic transcriptional programme through activation or repression of a variety of genes involved in the control of cell proliferation. The precise outcome of p53 activation depends on its localization, the amplitude of its stabilization, and the set of genes that it activates or represses, i.e. several parameters that Key words: acetylation, cancer, histone acetyltransferase (HAT), p53, post-translational modification, ubiquitylation. Abbreviations used: CREB, cAMP-response-element-binding protein; CBP, CREB-binding protein; HAT, histone acetyltransferase; Hdm2, human double minute 2; PCAF, p300/CREB-binding protein-associated factor. 1 To whom correspondence should be addressed (email [email protected]).

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are primarily controlled by a plethora of post-translational modifications of the p53 protein [4,5]. Importantly, since none of these modifications can be understood as being a simple on-off switch that governs a unique property of p53 [6], it is their combinatory effect that, like a barcode, determines the fate and transcriptional functions of p53 [7]. However, as shown below by the analysis of the crosstalk between two types of modification, ubiquitylation and acetylation, p53 modifiers target not only p53, but also other p53 modifiers. As a consequence, p53 activation must be rationalized in terms of activation of an integrated and intricate network of molecules that influence each other through multiple mechanisms, including post-translational modifications that have an impact on their stability, their interactions with various partners and their localization.

Main players in p53 ubiquitylation and acetylation Among the many types of post-translational modifications of p53, a large number (ubiquitylation, SUMOylation, NEDDylation, acetylation, methylation) target lysine residues [4]. This opens the possibility for direct competition or, conversely, synergism, between distinct p53 modifiers. Our recent work has led us to uncover novel functional connections between ubiquitylation and acetylation in the regulation of p53, adding complexity to the control of the ‘p53code’. Ubiquitylation is the covalent addition of ubiquitin polypeptides, either as monomers or as polyubiquitin chains, to lysine residues of a substrate [8]. This modification often targets the modified substrates for degradation by the 26S proteasome [9], but it may also serve many other functions, such as enzyme activation, endocytosis of membrane proteins or transcriptional regulation [10]. Biochem. Soc. Trans. (2010) 38, 98–103; doi:10.1042/BST0380098

Ubiquitin–Proteasome System, Dynamics and Targeting

Table 1 Ubiquitylation E3s within the p53 network

HAT given to the enzymes responsible for this modification), is an abundant post-translational modification involving the addition of an acetyl group to lysine residues. Four HATs Reference(s) play important roles in the regulation of p53: the structurally related p300 and CBP [CREB (cAMP-response-element[13,26,25,38] binding protein)-binding protein], and PCAF (p300/ CREB-binding protein-associated factor) and Tip60. They acetylate p53 on lysine(s) located at its C-terminus [30,31] (p300/CBP and PCAF [15]) or in its DNA-binding domain [39] (Tip60 [16]). Although there are some controversies about [40,41] the exact consequences of these modifications, it is generally [42] accepted that they are important for the transcriptional [43] functions of p53, as they favour its stabilization and its [37] sequence-specific DNA binding after DNA damage [4,15,17]. [44]

NEDD8, neural-precursor-cell-expressed developmentally down-regulated 8; Ub, ubiquitin. Protein

Target within the p53

Hdm2

network p53, PCAF, Tip60 (Ub) p53 (NEDD8)

p300/CBP PirH2 TOPORS

p53 p53 p53 (Ub) p53 (Sumo)

COP1 CHIP

p53 p53

ARF-BP1/Mule Cul4/DdB1 complexes CUL7

p53 p53, Hdm2 p53

E4F1 PCAF WWP1

p53 Hdm2 p53

[32] [33] [47]

CARP1/2 Synoviolin/Hrd1 SKP2

p53 p53 p300 (Ub-independent;

[48] [45] [27]

Parkin MKRN1

Ub?) p53 (Ub-independent) p53

[49] [50]

JFK TRIM24 MSL2

p53 p53 p53

[51] [52] [53]

E3 of viral origin E6/E6AP

p53

[54]

p53 p53

[55] [56]

E4orf6 ICP0

[46]

The specificity factors of the ubiquitylation reaction are the E3s (often called ubiquitin ligases), which generally promote (poly)ubiquitylation of the substrate by recruiting the latter via specific protein–protein interaction domains and presenting it to a ubiquitin-conjugating enzyme or E2 [8]. In the case of p53, many E3s have been described as being able to promote its ubiquitylation [11,12] (Table 1). Although the specific contribution of many of them in the global regulation of p53 remains to be clarified, possibly because they are involved in p53 regulation only under certain circumstances or in specific subcellular domains, the role of the RING finger oncoprotein Hdm2 (human double minute 2) [Mdm2 (murine double minute 2) in mice] as a central regulator of p53 stability is clearly established [13,14]. Indeed, Hdm2, even though its activity might not be always sufficient for efficient ubiquitylation of p53 and might require accessory E3 activities, is critical for ubiquitylation-dependent degradation of p53. Importantly, since the Hdm2 gene is itself a target for p53 transcriptional activity, activation of p53 leads to increased levels of Hdm2, thus forming a regulatory feedback loop. Acetylation, initially discovered as a modification of histone tails (hence the name histone acetyltransferase or

Cross-talk between ubiquitylation and acetylation in the regulation of p53 Over the years, many results have documented a complex interplay between acetylation and ubiquitylation in the control of protein fate [18]. Regarding p53 regulation, the HAT p300/CBP has been shown to be a strong antagonist of Hdm2, acting in two ways to stabilize and activate p53: first, it acetylates directly the same p53 residues that are ubiquitylated by Hdm2, and thus prevents p53 ubiquitylation by this E3 [19]; secondly, it inhibits Hdm2 ubiquitylation activity on p53 by direct acetylation of Hdm2 RING finger domain [20]. Reciprocally, Hdm2 can antagonize p300/CBP- and PCAF-dependent acetylation of p53, either directly by inhibition of their acetyltransferase activities [21–23], or indirectly by promoting the recruitment on p53 of the deacetylase HDAC1 [24]. Moreover, Hdm2 also mediates ubiquitylation and proteasome-dependent degradation of the HATs Tip60 [25] and PCAF [26]. Finally, recent results suggest that the F-box protein Skp2 (the specificity component of the ubiquitylation E3 SCFSkp2 ) can inhibit p300-mediated acetylation of p53, primarily by ubiquitylation-independent mechanisms [27]. Taken together, these results suggest a general trend for the respective roles of ubiquitylation and acetylation in p53 regulation: HATs are important for p53 stabilization and transcriptional activation, whereas ubiquitylation enzymes inhibit p53 by antagonizing acetylation and promoting degradation of p53. However, this simple model is contradicted by data showing that Hdm2 can interact with p300 and CBP, and that this interaction is important for Hdm2-dependent ubiquitylation and degradation of p53 [28,29]. Further work has provided a molecular explanation for this latter observation, by demonstrating that p300 [30] and CBP [31] are in fact bifunctional enzymes harbouring ubiquitylation E3 activity in addition to, and independently of, their well-known HAT activity. According to these data, p300/CBP E3 activity is required for efficient Hdm2dependent polyubiquitylation of p53 and its subsequent degradation by the proteasome. Whereas Hdm2 alone promotes only mono- or oligo-ubiquitylation of p53 (i.e. C The


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addition of single or short chains of ubiquitin on p53, a modification insufficient to support degradation by the proteasome), the combined action of Hdm2 and p300 allows conjugation of long polyubiquitin chains to p53, leading to efficient targeting of the protein to the proteasome, and to its rapid degradation [30]. The bifunctional nature of p300/CBP was the first illustration of a direct link between ubiquitylation and acetylation activities in the regulation of p53. In this case, the functional antagonism towards p53 activity (acetylation=activation, ubiquitylation=inhibition) is respected, which in fact places p300 and CBP as critical switches in p53 regulation, depending on which of their activities is promoted. Interestingly, each activity of p300 and CBP appears to be spatially controlled by differential nuclear and cytoplasmic mechanisms [31]. We have previously discovered other functional connections between ubiquitylation and acetylation within the p53 network, thanks to the identification of two novel ubiquitylation activities. Interestingly, instead of being inhibitory towards p53 activation, these activities promote its transcriptional functions [32,33]. The first observation was that the HAT PCAF, like p300 and CBP, is also a bifunctional enzyme with independent ubiquitylation and acetylation activities [33]. Similarly to p300/CBP, PCAF has no obvious ubiquitylation E3 domain, such as a RING or a HECT (homologous with E6-associated protein C-terminus) domain [8]. However, unlike p300/CBP, PCAF ubiquitylation activity does not target p53 directly, but instead Hdm2. This result has profound implications for our understanding of the regulation of the p53 network. First, it demonstrates the existence of yet another regulatory feedback loop within the network since, as mentioned above, PCAF is itself a substrate of Hdm2 [26]. Secondly, it challenges the well-accepted model that the regulatory loop between p53 and Hdm2 is controlled by Hdm2 autoubiquitylation activity. Hdm2 has indeed been shown to be able to auto-ubiquitylate itself in vitro, as do most E3s. Since its inactive mutant, in which the active-site cysteine residue (Cys464 in human) is changed to alanine, is much more stable than the wild-type protein when ectopically overexpressed in mammalian cells, it has been concluded that auto-ubiquitylation of Hdm2 was critical for its own stability, and thus for the regulation of p53 [34,35]. Although our data showing that PCAF is an E3 for Hdm2 do not exclude a possible contribution of auto-ubiquitylation in the control of Hdm2 stability, they clearly show that auto-ubiquitylation is certainly not the major actor in this process, since Hdm2 is strongly stabilized in the absence of PCAF [33]. Interestingly, this conclusion is reinforced by knockin experiments in mice showing that there is no significant difference in stability between wild-type Hdm2 and its active-site mutant, when expressed at physiological levels under the control of the endogenous promoter [36]. Indeed, by demonstrating that the increased stability of Hdm2 active-site mutant is an artefact probably linked to ectopic overexpression of the protein, and that consequently another ubiquitylation E3 must control C The


Hdm2 stability, these experiments indirectly support our conclusion that PCAF is the major ubiquitylation E3 for the control of Hdm2 stability. Thanks to its two activities, PCAF thus intervenes in a dual mode in the regulation of p53: it contributes to activation of p53 transcriptional activities via its HAT activity, whereas it promotes Hdm2 degradation and, consequently, p53 stabilization, via its ubiquitylation E3 activity. Therefore in contrast with p300/CBP whose HAT and ubiquitylation activities have opposite effects on p53 activation, the two activities of PCAF positively co-operate to favour stimulation of p53 functions. Our second observation, which adds to the notion of dense connections between ubiquitylation and acetylation in the control of p53, is the discovery that E4F1, an E1A-activated cellular transcription factor, is a ubiquitylation E3 for p53 [32]. Notably, E4F1 does not alter p53 stability. Instead, E4F1-dependent p53 ubiquitin conjugates are accumulated on the chromatin, and their presence in this structure correlates with the modulation of the transcriptional activities of p53, favouring recruitment of p53 on target genes that are involved in cell-cycle arrest at the expense of genes involved in apoptosis [32]. Very interestingly, the lysine residue of p53 targeted by E4F1 E3 activity is the same (Lys320 ) as the lysine residue acetylated by PCAF. This led us to propose that a direct competition between E4F1 and PCAF for Lys320 modification probably plays a critical role in the partitioning between the cell-cycle arrest and pro-apoptotic functions of p53 [32].

Sensitivity and robustness of p53 regulation: an emergent property of the network of p53 modifiers? Altogether, the functional connections described above between ubiquitylation and acetylation enzymes involved directly in the control of p53 (Figure 1) illustrate the complexity of the post-translational mechanisms used in mammalian cells to adequately regulate this key protein. Yet, these connections are only the tip of the iceberg, as (i) there is little doubt that additional cross-talk between ubiquitylation and acetylation will be discovered within the p53 network, and (ii) many other types of post-translational modification must be taken into account to understand the fine-tuning of the activity of the p53 network [4,6]. However, even though the final picture is far more complex, the crosstalk between ubiquitylation and acetylation highlights the intricacy of the network formed by p53 and its modifiers. They clearly show that activation of the p53 network by a given type of stimulus will not only translate into activation of a specific p53 modifier and then into a specific modification of p53 and a specific p53 barcode, but will also have an impact to various degrees on the other modifiers of the network. By analogy to the position of a ball within a fishnet that depends upon the different forces applied on the net, one can deduce from these observations that the status of p53


Figure 1 Schematic representation of the functional connections between ubiquitylation E3s and acetyltransferases within the p53 network Ac, acetylation; Ub, ubiquitylation. Blue and red represent modifications that favour and antagonize p53 activation respectively.

Furthermore, once the perturbation ceases, the equilibrium returns to its initial state. Thus the equilibrium is both robust and sensitive. Applied to p53 and its network of post-translational modifiers and modifications, this model of a metastable equilibrium fits well with the observed properties of the p53 response. First, in unstressed cells, the network remains stable, in an p53-latent state. However, a variety of signals, each leading to specific post-translational modification(s) of at least one member of the network, can displace the equilibrium towards a p53-activated state. Depending of the signal, and thanks to its rapid and specific propagation throughout the entire network, a new equilibrium can be rapidly reached, leading to a p53 response adapted to the signal. Finally, when the initial stress signal is stopped, the p53 network returns to its initial, p53-latent, state.

Conclusions

Figure 2 Schematic representation of the metastable equilibrium of the p53 network Modifications are indicated in blue; selected actors are indicated in red. ATM, ataxia telangiectasia mutated; CHK, checkpoint kinase.

Work of the last decade has highlighted that the critical tumour-suppressor role of the p53 pathway cannot be ascribed to p53 alone, as though this protein was endowed with super-anticancer power and could, alone, prevent tumour development. Instead, this pathway must be considered as an intricate network of p53 post-translational modifiers and interactors that has the ability to integrate multiple input signals and translate them into a specific cellular response, in a damage- and cell-dependent manner. Therefore a full understanding of p53 regulation will require not only a thorough description of all p53 modifications, but also the dissection of the functional and physical interconnections between its different modifiers. However, it is likely that the complexity of this system will resist reductionist approaches consisting of studying one by one the role(s) of each post-translational modification within the network, even though such studies are necessary. An important route to progress towards a better understanding of p53 might be to develop global approaches allowing us to describe the status (activity and post-translational modifications) of the main actors of the network after various stress stimuli. Such information should help us to understand how post-translational modifications propagate within the network, and thus to identify the key nodes and connecting lines that characterize this network at different stages of the p53 response.

Acknowledgement constantly depends upon a dynamic equilibrium determined by the interdependent effects of each modifier within the network (Figure 2). An interesting emergent property of such an equilibrium is its metastable character. The basal state, determined by the intrinsic properties of each component of the network, is stable as long as the properties of each component do not change. However, the equilibrium can be rapidly displaced by any modification introduced anywhere in the network.

We thank Dr L.K. Linares for her assistance in the preparation of Figure 2.

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Received 9 November 2009 doi:10.1042/BST0380098

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