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Laboratoire Jean-Claude Heuson de Cancérologie Mammaire, Institut Jules Bordet – Université Libre de Bruxelles, 127 boulevard de Waterloo, B-1000 Bruxelles, Belgium

(Requests for offprints should be addressed to M Lacroix; Email: Marc.Lacroix{at}ulb.ac.be)

	Abstract

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
p53 plays a key role in mediating cell response to various stresses, mainly by inducing or repressing a number of genes involved in cell cycle arrest, senescence, apoptosis, DNA repair, and angiogenesis. According to this important function, p53 activity is controlled in a very complex manner, including several auto-regulatory loops, through the intervention of dozens of modulator proteins (the ‘p53 interactome’). p53 mutations are observed in a significant minority of breast tumours. In the remaining cases, alterations of interactome components or target genes could contribute, to some extent, to reduce the ability of p53 to efficiently manage stress events. While the prognostic and predictive value of p53 is still debated, there is an increasing interest for p53-based therapies. The present paper aims to provide updated information on p53 regulation and function, with specific interest on its role in breast cancer.

	Introduction

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
While p53 seems to be dispensable for normal development (Donehower et al. 1992), it plays an important role in regulating cell fate in response to various stresses, either genotoxic (DNA alterations induced by irradiation, UV, carcinogens, cytotoxic drugs) or not (hypoxia, nucleotide depletion, oncogene activation, microtubule disruption, loss of normal cell contacts). The protein may be viewed as a node for the stress signals, which are then transduced, mainly through the ability of p53 to act as a transcription factor. p53 exerts its anti-proliferative action by inducing reversible or irreversible (senescence) cell cycle arrest, or apoptosis. It may also enhance DNA repair and inhibit angiogenesis.

Many types of stresses may be encountered during tumour development. The p53 function is often altered in cancer. It has been suggested that p53 could have evolved in higher organisms specifically to prevent tumour development (see notably in Vousden & Lu 2002). It is believed that this specific action is exerted mainly through the triggering of apoptosis (see notably in Haupt et al. 2003, Yu & Zhang 2005). Indeed, loss of p53 activity disrupts apoptosis and accelerates the appearance of tumours in transgenic mice (Attardi & Jacks 1999).

The qualitative and quantitative activity of p53 depends on its integrity (mutation status), its amount, and its specific posttranslational modifications induced by the activation of the different stress-induced signalling pathways. This leads to variable patterns of association between p53 and a number of other co-regulatory proteins, of which some may be tissue- or cell type-specific. Despite this complexity, p53 activity has been associated with prognosis and prediction of tumour response to various therapies and deserves further investigations with the perspective of developing more targeted treatments.

	Structure of p53

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
p53 is encoded by the Tp53 gene. Located at 17p13, this contains 11 exons spanning 20 kb. It belongs to a family of highly conserved genes that also includes TP63 and TP73, encoding p63 and p73 respectively.

Three functionally distinct regions have been identified in p53.

1. An acidic N-terminal region (codons 1–101), itself containing two major domains. (i) A transactivation acidic domain (codons 1–42). Codons 17–28 may interact with the ubiquitin ligase mouse double minute-2 homologue (MDM2), which plays a major role in p53 degradation (see below). Codons 22–26 (LWKLL) constitute an LXXLL-type co-activator recognition motif (Savkur & Burris 2004) involved in histone acetyltransferase P300 binding. It is believed that codons 11–27 may function as a secondary nuclear export signal (NES) and that DNA damage-induced phosphorylation may inhibit this activity. (ii) A proline-rich domain (codons 63–97) required for interaction with various proteins involved in the induction of apoptosis. It contains five PXXP motifs (PRMP at 64–67; PVAP at 72–75; PAAP at 77–80; PAAP at 82–85; PSWP at 89–92) that are involved in p53 interaction with P300 (Dornan et al. 2003). Interestingly, a polymorphism has been demonstrated at codon 72, where the proline is frequently replaced by an arginine. Both forms are morphologically wild-type and do not differ in their ability to bind to DNA in a sequence-specific manner. However, there are a number of differences between these p53 variants in their abilities to bind components of the transcriptional machinery, to activate transcription, to induce apoptosis, and to repress the transformation of primary cells (Thomas et al. 1999).
   
2. A central DNA-binding core region (codons 102–292). It recognizes a promoter consensus motif made of two 10 bp segments (RRRCWWGYYY) separated by 0–13 bp. This region is highly conserved throughout evolution. It is also the most homologous region among p53 family members (P63, P73).
   
3. A basic C-terminal region (codons 293–393), involved in tetramerization and regulation of p53 activity. It notably contains: (i) three nuclear localization signals (codons 305–322, 369–375, 379–384) recognized by a hetero-dimeric complex composed of importin alpha and beta that allows the p53 nuclear import (Fabbro & Henderson 2003); (ii) a tetramerization domain (codons 323–356), itself containing a primary NES (codons 339–352) recognized by the export receptor CRM1/exportin (Fabbro & Henderson 2003). p53 is active as a transcription factor only in the homotetrameric form. Tetramerization of p53 masks the primary NES and prevents export from the nucleus; (iii) a negative regulatory region (codons 363–393). By binding short non-specific DNA sequences, this region may prevent specific DNA binding to the core region (Weinberg et al. 2004).
   

	Genomic and non-genomic actions of p53

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
In normal cells not exposed to stress, the level and activity of p53 are very low. Upon stress, p53 is activated through a series of post-translational modifications and becomes able to bind to specific DNA sequences. The p53 recognition sequence is very loose and has been found in several hundred genes that are differentially modulated (induced or repressed) depending on the cell type, the nature of stress and the extent of damage. At low cellular levels, p53 modulates only a subset of the genes regulated at higher levels. The kinetics of target gene modulation may also vary.

In a study with a micro-array carrying 6000 capture sequences, 107 genes were found to be induced and 54 genes were repressed by p53 (Zhao et al. 2000). This result extrapolates to at least 500 up-regulated and 260 down-regulated p53 target genes.

Table 1, based on several papers (Yu et al. 1999, Vousden & Lu 2002, Liang & Pardee 2003, Nakamura 2004, Miled et al. 2005) lists a non-exhaustive series of p53-target genes that have been found to be altered by various stresses in many cell types.

Besides the regulation of apoptosis-related genes, p53 also appears to be able to act directly at the mitochondria. It can interact with BCL2 family members, such as the anti-apoptotic BCL2 itself and BCL-XL, and the pro-apoptotic BAK, thereby triggering mitochondrial outer membrane permeabilization and apoptosis (Schuler & Green 2005).

The quantitative, or even qualitative contribution of the direct, transcription-independent action to the global apoptotic activity of p53 has been debated. Observations such as the radio-resistant phenotype of the PUMA (BBC3)- and NOXA (PMAIP1)-knockout mice have been used as arguments against the general importance of transcription-independent mechanisms in vivo (Yu & Zhang 2005). It has also been observed that, in various cell lines, DNA damage induced by either ionizing radiation (IR) or topoisomerase inhibitors triggered a robust translocation of a fraction of p53 to mitochondria to a similar extent. Nevertheless, the cells succumbed to apoptosis only in response to topoisomerase inhibitors, but remained resistant to apoptosis induced by IR, suggesting that mitochondrial translocation of p53 does not per se lead to cell death (Essmann et al. 2005). Other investigators, by examining 179 mutant p53s, found no significant correlation between their apoptotic property and their ability to activate transcription of six p53-responsive genes (CDKN1A, MDM2, SFN, and the apoptosis-related BAX, p53AIP1, BBC3) (Kakudo et al. 2005). It is possible that rapid trans-activation-independent events could modulate the extent of apoptosis, which would however depend on transactivation-dependent events. However, recent observations suggest that the inverse could be true. Indeed, it has been shown that after genotoxic stress, the major regulator of apoptosis, BCL-XL, sequestered cytoplasmic p53. Nuclear p53 caused expression of PUMA, which then displaced p53 from BCL-XL, allowing p53 to induce mitochondrial permeabilization. Mutant BCL-XL that bound p53, but not PUMA, rendered cells resistant to p53-induced apoptosis irrespective of PUMA expression. These observations thus identify PUMA as the protein coupling the nuclear and cytoplasmic pro-apoptotic functions of p53 (Chipuk et al. 2005).

The central core region of p53 is of key importance in regulating apoptotic function, either transcription-dependent or -independent, as supported by the number of mutations affecting this region in apoptosis-deficient p53 cells. In addition to inducing genes that drive apoptosis, p53 can also activate the expression of genes that inhibit survival signalling (such as PTEN) or inhibit inhibitors of apoptosis (such as BIRC5) (Vousden & Lu 2002, Haupt et al. 2003, Meek 2004, Nakamura 2004, Lu 2005, Yu & Zhang 2005). Besides the central core, the proline-rich domain has been specifically associated with the apoptotic activity of p53 (Walker & Levine 1996). Deletion of this region leads to a complete loss of the apoptotic activity of p53. It could constitute an auxiliary protein-binding site and could be necessary for cellular cofactors specifically involved in the apoptotic activity of p53.

The p53-regulated genes that bring about senescence are less well characterized. However, CSPG2 has been strongly associated with senescence in prostate cancer cells (Schwarze et al. 2005).

	Biochemical modifications of p53

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
Posttranslational modification is a major mechanism regulating protein function. p53 may be phosphory-lated, cis/trans isomerized, acetylated, ubiquitinated, methylated, sumoylated, neddylated, glycosylated at multiple sites, reflecting its biological importance. This multisite modification, which exhibits a cell and tissue specificity and depends on the position in the cell cycle, is a complex regulatory program that fluctuates in response to cellular signalling triggered by DNA damage, proliferation and senescence, and thus appears as a dynamic ‘molecular barcodes’ (Yang 2005).

An overview of the p53 modifications that have been described to date is provided in Table 2. It is based on papers used for Table 1 and additional reports (Appella & Anderson 2001, Meek 2002, Bode & Dong 2004, Ou et al. 2005).

	Phosphorylation

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
p53 phosphorylation has been widely investigated. In most cases, it is associated with protein stabilization.

Three N-terminal sites, Ser15, Thr18, and Ser20, are particularly interesting because when phosphorylated, the interaction between p53 and its major negative regulator, MDM2, is diminished, while the binding of the acetyltransferase P300 is promoted, thereby increasing the level and stability of p53. Notably, Ser15 may be phosphorylated by IR (via ataxia-telangiectasia mutated; ATM) or UV (via ataxia-telangiectasia and Rad3-related; ATR). These stresses also lead to Ser20 phosphorylation, through the action of cell cycle checkpoint kinase 2 (CHK2) and CHK1 respectively. In fact, besides IR and UV, almost all stresses have been shown to induce Ser15 phosphorylation, which is thought to nucleate a series of subsequent p53 post-translational modifications (Meek 2004).

In some cases, p53 phosphorylation events are sequential. For instance, phosphorylation of Ser9 and Thr18 by CK1 is dependent of Ser6 and Ser15 phosphorylation respectively.

Another crucial N-terminal residue is Ser46. Its phosphorylation selectively promotes a p53 apoptotic response. Various kinases may be involved in this event, reflecting the activation of different stress pathways. For instance, HIPK2 mediates Ser46 phosphorylation in response to UV irradiation, although it seems that this alone is not sufficient to induce apoptosis. It has also been proposed that P38 can mediate the phosphorylation of Ser46 in response to UV. Neither P38 nor HIPK2 are involved in the Ser46 phosphorylation in response to IR, which requires both ATM and the p53-inducible gene, Tp53INP1, coding for p53DINP1. ATM does not directly phosphorylate p53, but it is likely to induce a kinase that might be co-activated by p53DINP1 to facilitate Ser46 phosphorylation (apoptosis-selective auto-regulatory loop) (Vousden & Lu 2002).

One important apoptosis-related protein, p53AIP1, is induced only when Ser46 is phosphorylated. Studies with the drug, etoposide, have confirmed that phosphorylation of p53 at Ser46 determines promoter selection and whether apoptosis is attenuated or amplified. High dose chemotherapy induced the phosphorylation of p53 on Ser46, whereas low dose chemotherapy did not. While Ser46-phosphorylated p53 targeted the promoter of the tumour suppressor PTEN in preference to MDM2 (thus abrogating the auto-regulatory loop that contributes to keeping the p53 level low), the inverse was observed in the absence of Ser46 phosphorylation. Accordingly, only high dose chemotherapy led to p53AIP1 induction, caspase 3 activation, and cell death (Mayo et al. 2005).

In addition to a common polymorphism at codon 72 (see below), p53 tumour also exhibits a rare single nucleotide polymorphism at residue 47. Wild-type p53 encodes proline at this residue, but in <5% of African Americans, this amino acid is serine. Notably, phosphorylation of the adjacent Ser46 by the proline-directed kinase P38 is known to greatly enhance the ability of p53 to induce apoptosis. The Ser47 polymorphic variant, which replaces the proline residue necessary for recognition by proline-directed kinases, is a markedly poorer substrate for phosphorylation on Ser46 by P38. Consistent with this finding, the Ser47 variant has an up to five-fold decreased ability to induce apoptosis compared with wild-type p53. This variant has a decreased ability to transactivate two p53 apoptotic target genes, p53AIP1 and BBC3, but not other p53 response genes; thus, the codon 47 polymorphism of p53 is functionally significant and may play a role in cancer risk, progression, and the efficacy of therapy (Li et al. 2005).

Experiments using p53 mutants with substitutions at Ser33, Ser46 or Thr81 have shown that phosphorylation of these sites (by P38 or Jun N-terminal kinase (JNK)) may independently lead to p53 stabilization, notably after exposure to UV (Appella & Anderson 2001).

In contrast to Ser315, Ser392 is phosphorylated only poorly after exposure of cells to IR, while it is strongly modified in response to UV (Appella & Anderson 2001).

In the C-terminal region of p53, phosphorylation of Ser315, Ser371, Ser376, Ser378, and Ser392 is well known. More recently, it has been shown that additional sites were also phosphorylated: Ser313, Ser314, Thr377, Ser378 (by both CHK1 and CHK2), Ser366 (by CHK2 only) and Thr387 (by CHK1 only). These events may alter the pattern of acetylation at Lys373 and Lys382, but not at Lys320, thus distinguishing between P300/CREB-binding protein (CBP) and P300/CBP-associated factor (PCAF) activity (see below) (Ou et al. 2005).

While most p53 phosphorylation events result in an increase in stability/activity of the protein, the phosphorylation of some sites (Thr55, Thr155, Ser215, Ser376) has been associated with enhanced p53 degradation. For instance, Thr55 can be phosphorylated by TAF_II250, the largest subunit of the general transcription factor TFIID, and this event enhances p53 degradation. Exposure of cells to UV decreases phosphorylation at Thr55 (Appella & Anderson 2001).

The COP9 signalosome (CSN) is an eight-subunit heteromeric complex that has homologies with the 26S proteasome bid complex. CSN has been reported to modulate ubiquitin ligase activity, as it directly interacts with cullin-domain ubiquitin ligases, catalyses deneddylation of these ligases, and is required for their proper function. Interestingly, CUL4A, a CSN-associated cullin-domain ubiquitin ligase has been shown to induce p53 degradation (see below). The CSN-associated kinases, CK2 and protein kinase D, are able to phosphorylate p53, and CK2 does so on Thr155. This dedicates p53 to rapid degradation by the ubiquitin–proteasome system. The importance of Thr155 is underlined by the fact that mutation of this residue is sufficient to stabilize p53 against human papilloma virus E6 oncoprotein-dependent degradation, which is mediated by E6AP, a ubiquitin ligase different from CUL4A. E6 is believed to play a major role in carcinoma of the cervix, where p53 mutations are rare.

Phosphorylation of Ser215 by the mitotic kinase serine/threonine protein kinase 15 (STK15) (also known as Aurora A) abrogates p53 DNA binding and transactivation activity (Liu et al. 2004b).

Ser376 (and Ser378) are constitutively phosphorylated by protein kinase C (PKC), which can contribute to p53 degradation (Chernov et al. 2001).

Not only the qualitative and quantitative pattern, but also the timing of p53 phosphorylation may vary depending on the stress. For instance, in response to IR increased phosphorylation of Ser6, Ser9, and Ser15 has been observed as early as 30 min after treatment, while exposure to UV induced a less-rapid, but more long-lived increase in the phosphorylation of these sites. This reflects the fact that ATR is more slowly activated than ATM (Appella & Anderson 2001).

	Dephosphorylation

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
In vitro dephosphorylation of p53 by the phosphatases PP1, PP2A, PP5, PPM1D and CDC14 has been shown. These may have different specificities, as shown, for instance, by the fact that PP1, but not PP2A, can dephosphorylate phospho-Ser15 (Haneda et al. 2004). PPM1D is of high interest, as it is induced by p53 and may dephosphorylate both p53 (at Ser15) and CHK1 (which may phosphorylate p53 at various sites) (Lu et al. 2006). Amplification of the PPM1D gene has been observed in breast cancer and seems to be associated with high aggressiveness (Rauta et al. 2006). Dephosphorylation of Ser376 by an ATM-regulated phosphatase allows 14-3-3 binding to phosphorylated Ser378, thereby contributing to p53 stabilization with consequent effects on site-specific DNA binding.

	Cis/trans isomerization

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
p53 activation involves a conformational change, brought about by cis/trans isomerization of certain proline residues by peptidyl-prolyl-cis-trans isomerase 1 (PIN1). PIN1 binds protein sites consisting of a phosphorylated serine or threonine followed by a proline; it then catalyses the isomerization of proline residues, which changes the conformation of p53. There are four Ser–Pro (Ser33–Pro34, Ser46–Pro47, Ser127–Pro128, Ser315–Pro316) and two Thr–Pro (Thr81–Pro82 and Thr150–Pro151) motifs on human p53 protein. Single mutations on these Ser–Pro or Thr–Pro sites do not lead to marked reduction of the p53-PIN1 interaction. However, a double point mutant (Ser33Ala, Ser315Ala) shows less binding to PIN1, and the triple point mutant (Ser33Ala, Ser315Ala, Thr81Ala) exhibits further reduced binding activity for PIN1, suggesting that these three sites are important for the p53-PIN1 interaction. It is possible that the Ser46–Pro47 site could also be involved in the process of cis/trans isomerization, considering the importance of Ser46 phosphorylation in p53 function. Whether the Ser127–Pro128 and Thr150–Pro151 motifs may be effectively targeted by PIN1 remains unknown at this time. The precise conformational changes induced by p53 due to different stress responses at different Ser–Pro or Thr–Pro sites are not yet clear. PIN1-induced conformational change in p53 inhibits the binding and/or stimulates the detachment of MDM2, leading to p53 stabilization. In addition, the conformational change may enhance the ability of P300 to acetylate p53 C-terminal lysines, and it may promote the binding of the p53 core domain to its specific promoter cognate sites, particularly those promoting apoptosis (Kohn & Pommier 2005).

Pro82 is essential for p53 interaction with CHK2 and consequent phosphorylation of Ser20 in response to DNA damage. These physical and functional interactions are regulated by PIN1. A sequence of events may thus be identified, in which phosphorylation of Thr81 allows PIN1 to isomerize p53, which further leads to p53-CHK2 interaction and phosphorylation of Ser20 (Berger et al. 2005).

	Acetylation

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
Acetylation has been shown to augment p53 DNA binding and to stimulate p53-mediated transactivation of target genes through the recruitment of co-activators. Acetylation is also thought to contribute to p53 stabilization by impairing ubiquitination of the acetylated residues. Intriguingly, while all evidence so far indicates that acetylation positively regulates p53 function (Brooks & Gu 2003), this modification seems also to regulate p53 subcellular localization, at least in part by activating its nuclear export (Kawaguchi et al. 2006).

P300, CBP and PCAF are ubiquitous transcriptional co-activators. They act as histone acetyltransferases, but may also acetylate various transcription factors, including p53. According to current data, P300/CBP may compete with MDM2 for binding to N-terminus of p53, so that a decrease in MDM2-p53 interaction associated with phosphorylation of N-terminal (especially Ser15) sites may favour P300/CBP binding and acetylation of Lys373 and Lys382. On the other hand, Ser15 phosphorylation is not absolutely required for p53 acetylation, as shown, for instance, by actinomycin D, which does not induce Ser15 phosphorylation but is a powerful agent in triggering p53 acetylation (Appella & Anderson 2001). Other p53 residues acetylated by P300/CBP are Lys370, Lys372 and Lys381. PCAF may acetylate Lys320.

It has been shown that upon non-apoptotic DNA damage such as that induced by cytostatic doses of cisplatin, PCAF acting in cooperation with homeo-domain-interacting protein kinase 2 (HIPK2) may acetylate p53. This HIPK2 action is independent of the Ser46 phosphorylation performed by the kinase upon severe genotoxic damage. Co-action of PCAF and HIPK2 selectively induce p53 transcriptional activity towards the CDKN1A promoter while depletion of either HIPK2 or PCAF abolishes this function. So, PCAF participates in the complex mechanisms allowing p53 to make a choice between growth arrest and apoptosis (Di Stefano et al. 2005). Interestingly, PCAF is a p53-induced gene (growth arrest-selective auto-regulatory loop) (Watts et al. 2004), while it is targeted for degradation (ubiquitinated) by MDM2 (Jin et al. 2004).

Experiments with histone deacetylase inhibitors on prostate cancer cells suggest that the acetylation of p53 at Lys373 is required for the p53-mediated induction of cell cycle arrest and apoptosis, while acetylation of p53 at Lys382 induces only cell cycle arrest (Roy et al. 2005).

The activation of p53 by P300/CBP can be achieved in a cooperative manner through the p53-binding proteins PRMT1 and CARM1 (co-activator-associated arginine methyltransferases). Whether p53 is a direct substrate for these two proteins is presently unknown.

	Deacetylation

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
It is likely that deacetylation provides a quick acting mechanism to stop p53 function once transcriptional activation of target genes is no longer needed. Deacetylation of p53 may be performed by multiple histone deacetylases (HDACs), at least by HDAC 1-3. The deacetylase sirtuin 1 (SIRT1) shows an in vitro activity on p53 peptides and it seems that cellular p53 is a major in vivo substrate of SIRT1 but not of the other six known SIRT proteins (SIRT 2-7) (Michishita et al. 2005). In fact, both HDAC1 and SIRT1 could be critical for p53-dependent stress response (Gu et al. 2004).

MTA2 (metastasis-associated protein 2)/PID (p53 target protein in the deacetylase complexes) specifically interacts with p53 both in vitro and in vivo, and its expression reduces significantly the steady-state levels of acetylated p53 by recruiting the HDAC1 complex. MTA2/PID expression strongly represses p53-dependent transcriptional activation, and, notably, it modulates p53-mediated cell growth arrest and apoptosis (Luo et al. 2000).

Numerous proteins modulating p53 activity have been shown to interfere with acetylation/deacetylation processes (not shown here).

	Ubiquitination

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
In normal cells, degradation is the only mechanism that abrogates all functions of p53, and this appears to be accomplished, in part, by the ubiquitin-26S proteasome system (the other way is ubiquitin-independent). The highly conserved protein, ubiquitin, targets substrate proteins for degradation by the 26S proteasome to peptides. Ubiquitin ligases realise the last step of ubiquitination. These enzymes exhibit a high level of target specificity.

In normal cells, the RING domain MDM2 is considered as the main ubiquitin ligase regulating the amount of p53. MDM2 binds to the N-terminal region and represses p53 activity via two mechanisms: by promoting p53 export to the cytoplasm and its consequent degradation and by blocking p53 transcriptional activation. The export of p53 requires an intact p53 NES. Several lysine residues located at the C-terminus of p53 may be MDM2-ubiquitinated: Lys370, Lys372, Lys373, Lys381, Lys382, Lys386 (Rodriguez et al. 2000). The ubiquitination of these lysine residues in the p53 C-terminus, including Lys305, is required to expose the NES even when p53 is bundled as a tetramer.

MDM2 is up-regulated by activated p53 and this generates a p53-MDM2 auto regulatory loop.

According to a current view, DNA damage leads to destabilization and accelerated degradation of MDM2. This limits MDM2 binding to p53 during the stress response and enables p53 to accumulate and remain active, even as p53 transcriptionally activates more MDM2. Thus, the induction of MDM2 RNA by activated p53 may create a reserve of MDM2 that can inactivate p53 once the DNA damage stimulus has abated and MDM2 is re-stabilized.

The physiological relevance of the p53-MDM2 loop is supported by various observations: (1) MDM2-knockout mice have an embryonic lethal phenotype (which can be abolished by the simultaneous inactivation of p53; (2) disruption of the p53-MDM2 interaction with synthetic competitive inhibitors is sufficient to induce a p53 response in cultured cells; (3) blocking MDM2 degradation via proteasome inhibition prevents p53 transactivation in DNA-damaged cells; (4) the activity of MDM2 is controlled by numerous factors and the p53-MDM2 loop is the focal point of the many different stresses that activate the p53 pathway (see below)

As many tumours inactivate wild-type p53 through MDM2 over-expression, exploiting the pathways that trigger MDM2 auto-degradation may be an important new strategy for chemotherapeutic intervention (Stommel & Wahl 2005).

COP1 (constitutive photomorphogenesis protein 1) is a RING domain ubiquitin ligase that inhibits p53-dependent transcription. Depletion of COP1 by short interfering RNA (siRNA) stabilizes p53 and arrests cells in the G₁ phase of the cell cycle. Over-expression of COP1 correlates with a striking decrease in steady state p53 protein levels and attenuation of the downstream target gene, CDKN1A, in cancers that retain a wild-type p53 gene status. Moreover, like MDM2, COP1 is a p53-inducible gene (Dornan et al. 2004).

The cytosolic chaperone-associated U-box domain ubiquitin ligase CHIP (C-terminus of hsc70-interacting protein) may induce the proteasomal degradation of p53. CHIP is thought to act in the quality control of protein folding, specifically ubiquitinating unfolded proteins associated with the molecular chaperones. CHIP-induced degradation has been observed for mutant p53, which was previously shown to associate with the chaperones Hsc70 and Hsp90, and for the wild-type form of the protein. Thus, mutant and wild-type p53 transiently associate with molecular chaperones and can be diverted onto a degradation pathway through this association (Esser et al. 2005).

The cullin-domain ubiquitin ligase CUL4A (cullin 4a) associates with MDM2 and p53, and ubiquitinates p53. Depletion of CUL4A leads to an accumulation of p53. CUL4A fails to increase the decay of p53 in mouse embryonic fibroblasts lacking MDM2. In addition, the CUL4A-mediated rapid decay of p53 is blocked by the MDM2 negative regulator p19^ARF(ARF for alternate reading frame). The results provide evidence for a cooperative role of CUL4A in the MDM2-mediated proteolysis of p53 (Nag et al. 2004).

The E6 oncoprotein of human papilloma viruses (HPVs) that are associated with cervical cancer utilizes the HECT domain ubiquitin ligase E6AP (E6-associated protein) to target p53 for degradation. In normal cells (i.e. in the absence of E6), p53 degradation is mediated by MDM2 rather than by E6AP. In HPV-positive cancer cells, the E6-dependent pathway of p53 degradation is not only active but, moreover, is required for degradation of p53, whereas the MDM2-dependent pathway is inactive. As the p53 pathway was reported to be functional in HPV-positive cancer cells, this finding indicates clearly that the ability of the E6 onco-protein to target p53 for degradation is required for the growth of HPV-positive cancer cells (Hengstermann et al. 2001).

Nuclear localization of p53 is essential for its tumour suppressor function. In contrast to most other ligases that act, or are believed to act in the nucleus, PARC (p53-associated parkin-like cytoplasmic protein), a RING domain ubiquitin ligase, directly interacts with p53 in the cytoplasm of unstressed cells. In the absence of stress, inactivation of PARC induces nuclear localization of endogenous p53 and activates p53-dependent apoptosis. Over-expression of PARC promotes cytoplasmic sequestration of ectopic p53. This suggests that PARC is a critical regulator in controlling p53 sub-cellular localization and subsequent function (Nikolaev et al. 2003).

PIRH2 (p53-induced protein, RING-H2 domain-containing) is a RING domain ubiquitin ligase that promotes p53 ubiquitination independently of MDM2. Expression of PIRH2 decreases the level of p53 protein and abrogation of endogenous PIRH2 expression increases the level of p53. Furthermore, PIRH2 represses p53 functions including p53-dependent trans-activation and growth inhibition. PIRH2, like MDM2 and COP1, participates in an auto-regulatory feedback loop that controls p53 function (Leng et al. 2003).

Using an osteosarcoma cell line, it was shown that TOPORS (topoisomerase I-binding arginine-serine-rich protein) could act on p53 as a RING finger-containing ubiquitin ligase. Over-expression of TOPORS was shown to result in a decrease in p53 protein expression (Rajendra et al. 2004). However, the exact role of TOPORS remains unclear, as it has also been shown to sumoylate p53, thereby abrogating its transcription activity. TOPORS was shown to associate with and stabilize p53, and to enhance the p53-dependent transcriptional activities of CDKN1A, MDM2 and BAX promoters. Over-expression of TOPORS consequently resulted in the suppression of cell growth by cell cycle arrest and/or by the induction of apoptosis (Lin et al. 2005).

Although P300 is known as an acetyltransferase, it has been suggested that it could cooperate with MDM2 to induce p53 polyubiquitination. In the presence of MDM2, P300 could poly-ubiquitinate the p53 residues mono-ubiquitinated by MDM2, thus contributing to p53 degradation; in the absence of MDM2, P300 might only act as a p53 acetyltransferase and therefore stimulates the transcriptional activity of p53 (Kohn & Pommier 2005).

Apparently, multiple degradation pathways are employed to ensure proper destruction of p53. How can one explain the apparent redundancy of ubiquitin ligases? A possibility is that ubiquitin ligases are expressed or act optimally in different cell or tissue types. It is also possible that one or more of these ubiquitin ligases are involved in the maintenance of p53 levels in the non-stressed or basal state, while others act only after a stress-induced p53 is produced. It appears likely that each of these ubiquitin ligases form protein complexes in the cell and the associated proteins may well differ for each of these ligases, connecting them to different regulatory circuits.

	Deubiquitination

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
USP7 (ubiquitin-specific protease 7, also known as HAUSP) has been shown to interact with p53, which can lead to p53 deubiquitination and stabilization. Its activity and global effect on p53 activity is, however, complex (see below).

	Ubiquitin-independent p53 degradation

Top Abstract Introduction Structure of p53 Genomic and non-genomic actions... Biochemical modifications of p53 Phosphorylation Dephosphorylation Cis/trans isomerization Acetylation Deacetylation Ubiquitination Deubiquitination Ubiquitin-independent p53... Sumoylation Methylation Neddylation ADP-ribosylation O-glycosylation Modulators of p53 activity The key role of... Interactions between p53 and... Mechanisms of p53 apoptosis... Other interactors modulating the... Auto-regulatory loops in p53... Mechanisms for loss of... p53 alterations, breast tumour... p53 alterations and response... Tumours Additional comments on p53... p53 pathway-based therapies General conclusion References

 
The proteasomal degradation of p53 is regulated by both (poly) ubiquitination, targeting p53 for degradation by the 26S proteasome and by a MDM2- and ubiquitin-independent process. This appears to be mediated by the core 20S catalytic chamber of the 26S proteasome and is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). NQO1 physically interacts with p53 in an NADH-dependent manner and protects it from 20S proteasomal degradation. Remarkably, the vast majority of NQO1 in cells is found in physical association with the 20S proteasomes, suggesting that NQO1 functions as a gatekeeper for these 20S proteasomes. By competing with NADH, NQO1 inhibitors including dicoumarol and various other coumarins and flavones induce ubiquitin-independent proteasomal p53 degradation and thus inhibit p53-induced apoptosis.

The NQO1 pathway plays a role in p53 accumulation in response to IR, as co-expression of NQO1-specific siRNA with p53 prevented the accumulation of the latter following IR. Escaping MDM2-mediated degradation is probably not sufficient for efficient p53 stabilization following IR, because p53 is still susceptible to 20S proteasomal degradation. In order to achieve efficient p53 accumulation following irradiation, NQO1-p53 interaction could be increased to eliminate p53 degradation by the 20S proteasomes. NQO1 might notably play a role in p53 accumulation under oxidative stress. Reactive oxygen species (ROS) are known to induce NQO1, which, in turn, reduces ROS. The ability of NQO1 to support p53 accumulation following oxidative stress may contribute to cellular defence mechanisms against ROS.

The core 20S proteasomes are abundant and ubiquitously present in the cells. They have been widely regarded as being incapable of degrading folded proteins and are therefore considered to be latent proteasomes. Degradation studies with natively unfolded proteins suggest that unstructured proteins might have an intrinsic capacity to enter the pore of the 20S proteasome. Furthermore, the unstructured protein even when flanked with well-structured regions is still susceptible to 20S proteasomal degradation. Therefore, a common feature of ubiquitin-independent and 20S proteasomal degraded proteins could be the presence of an unstructured protein region. Indeed, both the N-and the C-terminal regions of p53 have been identified as unstructured regions and could facilitate p53 degradation by the 20S proteasomes. p53 could be inherently unstable and degraded ‘by default’ by the 20S proteasome, unless stabilized by a molecule like NQO1. p53, when engaged in a large functional complex could be protected from 20S proteasomal degradation as a consequence