Regulation of Mdmx and its role in the p53 pathway

Regulation of Mdmx and its role in the p53 pathway

Meulmeester, Erik

Citation

Meulmeester, E. (2006, February 2). Regulation of Mdmx and its role in the p53 pathway.

Retrieved from https://hdl.handle.net/1887/4280

Version:

Corrected Publisher’s Version

License:

Licence agreement concerning inclusion of doctoral thesis in the

_{Institutional Repository of the University of Leiden}

Downloaded from:

https://hdl.handle.net/1887/4280

Regulation of Mdmx and its role in the p53 pathway.

Proefschrift

ter verkrijging van

de graad van Doctor aan de Universiteit Leiden,

op gezag van de Rector Magnificus Dr.D.D.Breimer,

hoogleraar in de faculteit der Wiskunde en

Natuurwetenschappen en die der Geneeskunde,

volgens besluit van het College voor Promoties

Promotiecommissie

Promotor:

Prof. Dr. P. ten Dijke

Co-promotor:

Dr. A.G. Jochemsen

Referent:

Dr. A. Shvarts (Universiteit Utrecht)

Overige Leden:

Prof. Dr. R. Bernards (Universiteit Utrecht en

Nederlands Kanker Instituut)

Prof. Dr. L Mullenders

Printing: Febodruk BV, Enschede, The Netherlands. Printing of this thesis was

financially supported by the Juriaanse stichting and the Dutch Cancer Society

(KWF)

The research presented in this thesis was performed at the department of

Molecular Cell Biology, Leiden University Medical Centre, Leiden, The

Netherlands. This work was supported by a grant from the Dutch cancer

society (KWF).

Contents

Chapter

1

Introduction 7

Chapter 2

Critical Role for a Central Part of Mdm2 in

23

the Ubiquitylation of p53

Chapter 3

Amplification of Mdmx (or Mdm4) Directly

37

Contributes to Tumor Formation by Inhibiting

p53 Tumor Suppressor Activity

Chapter 4

Loss of HAUSP-Mediated Deubiquitination

53

Contributes to DNA Damage-Induced

Destabilization of Hdmx and Hdm2

Chapter 5

Modification of Mdmx by SUMO-1 and

75

Chapter 1

Introduction

Published in modified form in:

Cell cycle (2005) 9:n/a

Introduction

Abstract. The p53 tumor suppressor protein has a major role in protecting genome integrity. Under

normal circumstances Mdmx and Mdm2 control the activity of p53. Both proteins inhibit the transcriptional regulation by p53, while Mdm2 also functions as an E3 ubiquitin ligase to target both p53 and Mdmx for proteasomal degradation. HAUSP counteracts the destabilizing effect of Mdm2 by direct deubiquitination of p53. Subsequently, HAUSP was shown to deubiquitinate Mdm2 and Mdmx, thereby stabilizing these proteins. The ATM protein kinase is a key regulator of the p53 pathway in response to double strand breaks (DSBs) in the DNA. ATM fine-tunes p53's response to DNA damage by direct phosphorylation, by regulating additional post-translational modifications of this protein, and by affecting two p53 regulators: Mdm2 and Mdmx. ATM directly and indirectly induces Mdm2 and Mdmx phosphorylation, resulting in decreased activity and stability of these proteins. Recently a mechanism was provided for the reduced stability of Mdm2 and Mdmx by showing that ATM-dependent phosphorylation lowers their affinity for the deubiquitinating enzyme HAUSP. Altogether, the emerging picture portrays an elaborate, but fine-tuned, ATM-mediated control of p53 activation and stabilization following DNA damage. Further insight into the mechanism by which ATM switches the interactions between HAUSP, Mdmx, Mdm2 and p53, to favor p53 activation may offer new tools for therapeutic intervention in the p53 pathway for cancer treatment.

The p53 pathway

The p53 tumor suppressor gene encodes a sequence-specific transcription factor whose activity is either disabled or attenuated in the vast majority of human cancers (Vogelstein et al., 2000; Michael and Oren, 2002). Its inactivation occurs in about 50% of human tumors through mutations affecting the p53 locus directly (Hollstein et al., 1991). p53 transcriptionally activates a vast, constantly growing number of target genes, resulting in various biological outcomes such as cell-cycle arrest and apoptosis (Vousden, 2000; Michael and Oren, 2002; Vogelstein et al., 2000). Several types of stress, such as oncogene activation, hypoxia and DNA damage, result in an increase in p53 levels and the subsequent activation of p53 target genes (Vogelstein et al., 2000). One of the best-characterized target genes of p53 is the mdm2 gene, which contains two promoters. The first promoter (P1) drives mdm2 expression constitutively (Jones et al., 1996), while p53 binds two adjacent p53-responsive elements within the second promoter (P2), thereby promoting transcription of the mdm2 gene (Momand et al., 2000; Barak et al., 1993; Prives, 1998). Under normal circumstances, p53 is tightly regulated through the interaction with its negative regulator Mdm2, which counteracts p53 function in a number of ways (Momand et al., 2000; Barak et al., 1993; Prives, 1998; Minsky and Oren, 2004). The autoregulatory negative feedback loop, whereby p53 induces Mdm2 expression resulting in the repression of p53

function, most probably serves as an important mechanism to restrain p53 activity in normal cells. Therefore, uncontrolled, high expression of Mdm2 may result in improper inactivation of p53 function. It has been shown that in 5-10% of all human tumors Mdm2 is overexpressed, due to gene amplification, transcriptional- or post-transcriptional mechanisms (Momand et al., 2000; Juven-Gershon and Oren, 1999). In most of these cases the p53 gene is wild type, presumably because Mdm2 overexpression alleviates the selective pressure for direct mutational inactivation of the p53 gene.

Mdmx, a homologue of Mdm2

The Mdmx protein was originally identified in a screen for p53-binding proteins (Shvarts et al., 1996). Subsequently, Mdmx was also recognized as an Mdm2 binding protein in a yeast two-hybrid screen (Tanimura et al., 1999; Sharp et al., 1999). Co-immunoprecipitation experiments confirmed complex formation of Mdmx with p53 and Mdm2 (Stad et al., 2000). It must be noted that the hetero-oligomerization between Mdmx and Mdm2 was much stronger than the homo-typic interaction of Mdmx or Mdm2 (Tanimura et al., 1999). Comparison of the Mdmx and Mdm2 proteins revealed their strong structural homology (figure 1). The most conserved regions of Mdmx and Mdm2 comprise the N-terminal p53-binding domain, a zinc-binding motif and a C-terminal RING-finger (Shvarts et al., 1996). The similar

Figure 1. Protein structures of Mdmx and Mdm2. Percentage identity and similarity between domains in Mdmx and Mdm2 are given in percentages. Depicted domains: p53-binding domain (p53-BD), nucleolar localization signal (NLS), nuclear export signal (NES), Zinc finger (ZN-finger), RING finger and nucleolus localization signal (NoLS).

requirements for Mdmx and Mdm2 to interact with p53 emphasized the resemblance of the p53-binding domains (Bottger et al., 1999). The interaction between Mdmx and Mdm2 is mediated through the C-terminal RING-finger domains, while the function for the zinc-finger motifs is largely unknown. Incontrast to Mdm2, Mdmx contains no functional nuclear localization signal(NLS) or nuclear export signal (NES) (Li et al., 2002a; Migliorini et al., 2002; Gu et al., 2002). The acidic region(residues 237 to 274) is less well conserved at the primary amino acid level between the two proteins, although Mdmx alsocontains many acidic amino acids in the same region. Due tothe lack of an NLS or NES, the subcellular localization of the Mdmx protein is determined by its association with other cellularproteins, providing a level of regulation of Mdmx activity.

The first reported activity of Mdmx is the inhibition of p53-mediated transcription activation as measured with p53-reporter luciferase constructs, as well as of endogenous p53-targets (Shvarts et al., 1996). Accordingly, knocking down Mdmx expression by RNAi increased the expression of the p53 target gene p21 and resulted in a p53-dependent growth inhibition (Danovi et al., 2004). The inhibition of p53-mediated transcription activation by Mdmx is dependent on its p53-binding domain (Shvarts et al., 1996). A possible mechanism for p53 inactivation is the inhibition of p300/CBP-mediated acetylation of p53 by Mdmx (Sabbatini and McCormick, 2002; Danovi et al., 2004). The acetylation of p53 by p300/CBP is well accepted to enhance p53-mediated transactivation (Avantaggiati et al., 1997; Lill et al., 1997; Scolnick et al., 1997; Gu and Roeder, 1997). Since p300 and Mdm2/Mdmx have an

overlapping binding site on p53, it has been suggested that Mdm2/Mdmx may block p53 acetylation by preventing p300 binding to p53 (Finlan and Hupp, 2004; Danovi et al., 2004). The role of Mdmx in regulating the p53 acetylation status was confirmed with the use of

mdmx heterozygous and homozygous knockout

cells (Danovi et al., 2004). Concomitantly with decreased Mdmx levels in these cells, the p53 acetylation increased. Mdm2 not only prevents the acetylation of p53 but has also been implicated to deacetylate p53 by active recruitment of HDAC1 (Ito et al., 2002). Although Mdmx also associates with HDAC1 it is unlikely that the main mechanism of p53 transcriptional inhibition by Mdmx is dependent on HDAC1. Especially since the p53-binding domain of Mdmx is unable to bind HDAC1, but is sufficient to prevent p300-mediated p53 acetylation (Danovi et al., 2004). Nevertheless, it cannot be excluded that under certain circumstances Mdmx recruits HDAC1 to deacetylate p53.

Although the physiological function for Mdmx in the regulation of p53 has been a matter of debate, the importance of Mdmx in p53-regulation has been highlighted by the fact that the absence of Mdmx causes p53-dependent embryonic lethality (Parant et al., 2001; Finch et al., 2002; Migliorini et al., 2002). The mdmx homozygous knockout embryos are characterized by a retarded growth ability and massive p53-induced apoptosis in the neuroepithelium (Finch et al., 2002; Migliorini et al., 2002). Mdmx-null embryos are reduced in size and die at midgestation (E7.5-E12). The expression of the cyclin dependent kinase p21, a p53 inducible gene, was strongly upregulated in both Mdmx-null embryos and mouse embryo

fibroblasts (MEFs) (Parant et al., 2001; Migliorini et al., 2002). Interestingly, it was reported that deletion of p21 delays the midgestation lethality observed in Mdmx-null mice (until E15.5) (Steinman et al., 2004). These results suggest that Mdmx suppresses p53-mediated cell growth during development. Since

mdm2- knockout mice are also embryonic lethal

in a p53-dependent manner (Montes de Oca Luna et al., 1995; Jones et al., 1995), Mdmx and Mdm2 cannot substitute for one another, at least in early embryonic life. These results suggest that Mdmx and Mdm2 have non-redundant functions.

Further evidence for an important role of Mdmx in p53 regulation was revealed studying a large series of gliomas. The Mdmx gene was found to be the common amplified gene in a subset of gliomas (Riemenschneider et al., 1999; Riemenschneider et al., 2003). In addition, Mdmx was found overexpressed and/or expressed into alternative proteins in about 30% of human tumor cell lines, in general correlating with the presence of wild-type p53 (Ramos et al., 2001). More recently, it was found that Mdmx is overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53 (Danovi et al., 2004). In addition, Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with H-rasV12. These results indicate that Mdmx is an important regulator of p53 that, upon overexpression, overcomes the necessity for p53 mutation or deletions in human tumors. The fundamental research on Mdm2 and Mdmx as negative regulators of p53 has benefited the possible therapeutic treatment of cancer by generating drugs to inhibit their interaction with p53. Small molecule inhibitors, like RITA and Nutlin-3 impair the Mdm2-p53 interaction, thereby activating p53 (Vassilev, 2004; Issaeva et al., 2004). These drugs probably also inhibit the Mdmx-p53 interaction which may contribute to the observed growth inhibition of tumors.

The Mdmx, Mdm2 and p53 loop.

As discussed above, both Mdmx and Mdm2 can inhibit the transcriptional activity by p53 (figure 2). In addition, Mdm2 targets p53 for ubiquitin-dependent proteasomal degradation

Figure 2. Mdm2 and Mdmx inhibit the function of p53. Cell cycle arrest and apoptosis genes are transcriptionally activated by p53. Both Mdm2 and Mdmx inhibit the transcriptional activity of p53. The

mdm2 gene is activated by p53 (dashed line) resulting

in a negative feedback loop. Mdmx is proposed to enhance Mdm2 in the ubiquitin dependent degradation of p53. Mdm2 mediates the degradation of both p53 and Mdmx.

(Kubbutat et al., 1997; Honda et al., 1997; Haupt et al., 1997). With the use of recombinant proteins in an in vitro assay it was suggested that Mdm2 does not attach poly-ubiquitin chains to p53, but rather mediates multiple mono-ubiquitination (Lai et al., 2001). However, others showed that high levels of Mdm2 are able to poly-ubiquitinate p53 resulting in the degradation of p53 in the nucleus (Li et al., 2003; Xirodimas et al., 2001). In contrast, low levels of Mdm2 were only able to mono-ubiquitinate p53, which was proposed to be required for nuclear export of p53 (Geyer et al., 2000; Boyd et al., 2000; Li et al., 2003). One could pose the question why p53 is exported from the nucleus to the cytoplasm, while it can also be degraded in the nucleus. One possibility may be that low levels of Mdm2 initially transfer p53 to the cytoplasm, to hamper its nuclear functions, which is followed by the cytoplasmic degradation of p53.

The finding that Mdm2/p300 complexes participate in the degradation of p53 suggested a role for p300 in p53 turnover (Grossman et al., 1998). This finding was supported by the observation that mutants of Mdm2 unable to bind p300 were still able to ubiquitinate p53, but lacked the ability to degrade p53 (Argentini et al., 2001; Zhu et al., 2001). A possible

mechanism for p300-mediated degradation of p53 was provided by the observation that p300 might function as an E4 ubiquitin ligase (Grossman et al., 2003). It was proposed that p300 positively regulates p53 activity at low levels of Mdm2, however, at high levels of Mdm2, p300 might act as a negative regulator of p53 (Kawai et al., 2001; Yuan et al., 1999). A simple model for this apparent contradictory role for p300, but consistent with the current data, is that under physiological conditions p300 exists in a trimeric complex with p53 and Mdm2. This complex targets p53 for degradation, in which Mdm2 mono-ubiquitinates and p300 poly-ubiquitinates p53 (Grossman et al., 2003). However, upon various kinds of stress the ubiquitin ligase activity of Mdm2 is impaired by ATM and c-Abl-mediated phosphorylation (Goldberg et al., 2002; Maya et al., 2001). In this scenario p300 is unable to poly-ubiquitinate p53 due to the absence of mono-ubiquitinated p53. In this model p300 senses p53 mono-ubiquitination, which allows p300-mediated poly-ubiquitination of p53, while in the absence of mono-ubiquitination p300 could positively regulate p53 functions.

The regulation of p53 degradation was revealed to be even more complex by the finding that besides a functional RING finger also an internal domain of Mdm2 is required for ubiquitination of p53 (Meulmeester et al., 2003; Kawai et al., 2003b). Although it had been reported before that deletions within this domain disrupt p300 binding, coinciding with retarded p53 degradation, we find that disruption of this domain also diminishes p53 ubiquitination. Interestingly, the RING finger and the acidic domain can cooperate, even when located on two different proteins, resulting in proper p53 ubiquitination and degradation. These results suggest that yet another cofactor might be required for p53 ubiquitination and degradation. One such factor could be HDAC1 that was proposed to facilitate Mdm2-mediated ubiquitination of p53, by deacetylating the required lysine residues. Thus, Mdm2 inhibits p53 activation by binding its transcriptional activation domain and mediating its proteasomal degradation. The finding that Mdm2 can also ubiquitinate histones around p53 responsive promoters suggests a new mechanism of inhibiting p53 activity (Minsky and Oren, 2004). Recently, two other E3 ubiquitin ligases for p53 have been described. In the laboratory of S.

Benchimol the p53-induced gene pirh2 gene was found, which encodes a RING-H2 domain-containing protein with intrinsic ubiquitination activity (Leng et al., 2003). The Pirh2 protein interacts with and ubiquitinates p53, in an Mdm2-independent manner, resulting in the degradation of p53 (Leng et al., 2003). Pirh2, therefore, forms a negative auto-regulatory feedback loop with p53, like Mdm2. About a year later COP1 was also found to ubiquitinate and degrade p53, independent of Pirh2 and Mdm2 (Dornan et al., 2004). The physiological relevance for the negative regulators Pirh2 and COP1 requires further research, especially since both proteins are unable to rescue the embryonic lethality of mdm2- or mdmx- knockout mice. It could be hypothesized that COP1 and/or Pirh2 regulate p53 in specific stress responses or at specific stages in development.

The role of Mdmx in p53 degradation is at the moment difficult to understand. Initial studies suggested that high levels of Mdmx could stabilize p53 by blocking nuclear export of ubiquitinated p53, resulting in the accumulation of ubiquitinated p53 in the nucleus (Jackson and Berberich, 2000; Stad et al., 2001; Stad et al., 2000). Since Mdmx has no detectable E3 ubiquitin ligase activity in cells, these results suggest that Mdm2's ability to ubiquitinate p53 is not impaired. It was even found that Mdmx could act as a bridge between p53 and an Mdm2-mutant lacking the p53-binding domain, resulting in ubiquitinated p53 (Stad and Jochemsen, unpublished observations). Since overexpression of Mdmx elevates p53 stability and ubiquitination, a role of Mdmx at the post-ubiquitination stage in p53 degradation would be suggested. It could be hypothesized that overexpression of Mdmx interferes with formation of Mdm2/hRad23 complexes. The hRad23 protein is a bridge between ubiquitinated proteins and the proteasome and when bound to Mdm2 the degradation of p53 is enhanced (Glockzin et al., 2003; Brignone et al., 2004). Transfection studies have also suggested a role for Mdmx in the stabilization of Mdm2 (Stad et al., 2001; Stad et al., 2000). In contrast to p53 stabilization, Mdmx-mediated stabilization of Mdm2 is accompanied by a decrease in poly-ubiquitinated forms of Mdm2 (Stad et al., 2001). These results suggest a possible role for Mdmx in maintaining a pool of inactive Mdm2, which can be activated under specific conditions. As expected from the results above, RNAi-mediated knock down of Mdmx resulted in a decrease of

Figure 3. The complex web of ATM-mediated activation of the p53 pathway. ATM mediates direct and indirect phosphorylation of p53, while 14-3-3 binding to p53 is augmented by ATM-mediated de-phosphorylation of p53. Phosphorylation of Strap by ATM results in the recruitment of Strap/p300 complexes towards p53 that elevates its acetylation. A safeguard mechanism exists to ensure proper p53 activation by inhibiting its inhibitors Mdm2 and Mdmx. Phosphorylation of Mdmx/Mdm2 attenuates their interaction with the ubiquitin protease HAUSP, resulting in the instability of Mdmx and Mdm2. Thus ATM activates p53 via a sophisticated mechanism, while it ensures proper activation by inhibition of its negative regulators

Mdm2 levels in both U2OS (wt p53) and H1299 (p53 null) cells, and consequently an increase in p53 levels (U2OS cells) (Gu et al., 2002). However, when the identical RNAi construct was introduced into MCF7 cells, neither Mdm2 nor p53 levels were altered (Kawai et al., 2003a). A careful titration of Mdmx revealed that low levels of Mdmx can enhance Mdm2-mediated degradation of p53, although high levels of Mdmx stabilize p53 (Gu et al., 2002). The finding that Mdmx does facilitate Mdm2-mediated ubiquitination of p53 in vitro supported this model (Linares et al., 2003). These authors mention that the knock down of Mdmx results in an increase of both Mdm2 and p53. In contrast to Kawai et al. (2003a), we have not been able to detect a significant effect on p53 or Mdm2 levels upon RNAi-mediated knock down of Mdmx in MCF-7 cells, (Danovi et al., 2004). However, we detected an increase in p53 acetylation levels, reflecting activation of p53, when extracts from

mdmx- deficient embryos were compared to

heterozygous or wild type embryos. Moreover, we observed an increase in p53 responsive genes in mdmx-knockout embryos, while only a marginal increase in p53 levels was detected (Danovi et al., 2004). Although it is well

accepted that Mdmx is a negative regulator of p53, the mechanism of its function remains obscure. Further research is required to address the question how Mdmx inhibits p53 activity. Although RNAi is a powerful tool to investigate the effect of individual proteins in cells, it must be taken into account that off-target effects are well known to occur, like activation of an interferon response (Sledz et al., 2003). Secondly, decreasing the amount of Mdmx may have an impact on both p53 activity, and subsequently on mdm2 gene transcription, and Mdm2 protein stability, resulting in mixed signaling. The use of Mdm2 mutants unable to bind Mdmx, while retaining its E3 ligase activity, could be a useful tool to address the mechanism by which Mdmx enhances the degradation of p53.

p53 activation upon double strand DNA breaks.

Upon DNA damage a complex network of responses is initiated (Bakkenist and Kastan, 2004; Shiloh and Lehmann, 2004). One branch of it recognizes and repairs the damaged DNA, while many other pathways activate numerous

other responses related to many aspects of cellular metabolism. A prominent response is activation of the damage-induced cell cycle checkpoints (Lukas et al., 2004). Activation and stabilization of the p53 protein plays an important role in one of the major pathways that control the G1/S checkpoint. Double strand breaks (DSBs) evoke the DNA damage response to its fullest extent (Shiloh, 2003; Bassing and Alt, 2004). The primary regulator of the DSB response is the nuclear protein kinase ATM. DSBs induce rapid activation of ATM which involves its auto-phosphorylation (Bakkenist and Kastan, 2003). Upon activation, ATM rapidly phosphorylates numerous substrates, thereby modulating their activity or stability and affecting the pathways in which they function (Shiloh, 2003; Kurz and Lees-Miller, 2004). In this introduction the focus will be on the ATM-mediated activation of the p53 pathway (figure 3). In normal growing, untransformed cells p53 has a short half-life of about 30 minutes. Following DSBs, p53 becomes rapidly stabilized and functionally activated, as a consequence of several post-translational modifications. The p53 protein plays a major role in cellular stress responses by initiating an important protective mechanism to allow for the repair of DNA damage or to remove cells from the population by means of apoptosis. Activated ATM rapidly phosphorylates p53 on Ser15, and this phosphorylation was reported to enhance its transcriptional activity (Banin et al., 1998; Lambert et al., 1998). The ATM-Related kinase, ATR, maintains phosphorylation of Ser15 at later stages during the DNA damage response. Upon Ser15 phosphorylation, p53 is phosphorylated on Thr18 by CK1, which might affect the Mdm2/p53 interaction (Sakaguchi et al., 2000; Schon et al., 2002; Saito et al., 2003). Furthermore, ATM (and later, probably ATR) orchestrates the phosphorylation of p53 on Ser20, which was reported to increase affinity for the transcriptional co-activator p300 (Dornan and Hupp, 2001; Dumaz and Meek, 1999). Not only that, ATM further controls the phosphorylation of p53 on several other residues indirectly by activating several kinases, such as Chk1, Chk2 and HIPK2 (Gatei et al., 2003; Matsuoka et al., 1998; Shieh et al., 2000; Chehab et al., 2000; D'Orazi et al., 2002; Saito et al., 2002). Interestingly, upon DNA damage the p53 protein is also subjected to de-phosphorylation on Ser376 in an ATM-dependent fashion (Waterman et al., 1998). The de-phosphorylation

of Ser376 creates a consensus 14-3-3 binding site and leads to the association of p53 with 14-3-3. This, in turn, elevates p53's sequence-specific DNA binding. The phosphorylation of p53 co-factors is yet another way by which the ATM kinase stimulates the activation of p53. It has been reported that the co-factor Strap is phosphorylated by ATM on Ser203, resulting in increased nuclear localization and association with p300 (Demonacos et al., 2004). Phosphorylation of Strap results in increased acetylation and activation of p53, suggesting that Strap/p300 complexes are recruited to p53, to induce its acetylation. In conclusion, ATM regulates many post-translational modifications of p53 ensuing a proper p53 response to DSBs. Besides phosphorylation and acetylation, the p53 protein is also methylated, NEDDylated, SUMOylated and modified by Pin1 upon DNA damage (Chuikov et al., 2004; Xirodimas et al., 2004; Gostissa et al., 1999; Rodriguez et al., 1999; Zacchi et al., 2002; Zheng et al., 2002). It would be very interesting to find out whether ATM-mediated signaling also influences the efficiency of these p53 modifications upon DNA damage.

Degradation of Mdmx and Mdm2: an important step in p53 activation.

ATM not only coordinates modification of p53 but also influences the cellular abundance of Mdm2 and Mdmx, which is now emerging as an important step in the p53 activation (Stommel and Wahl, 2004; Pereg et al., 2005). The ATM-mediated phosphorylation of Ser395 on Mdm2 was shown to affect the nuclear export and degradation rate of p53. In addition, ATM mediates indirect phosphorylation of Mdm2 on Tyr394 via the c-Abl kinase (Shafman et al., 1997; Baskaran et al., 1997; Goldberg et al., 2002). It has been suggested that the phosphorylation of Mdm2 by c-Abl reduces the Mdm2-mediated ubiquitination and nuclear export of p53 (Goldberg et al., 2002). Lastly, Okamoto and coworkers have shown that ATR directly phosphorylates Mdm2 on Ser407 in response to replication block, and this phosphorylation was proposed to reduce the ability of Mdm2 to degrade p53 after DNA damage (Shinozaki et al., 2003). These results could, at least partly, explain the impaired degradation and inhibition of p53 activity by Mdm2 upon DNA damage. Further explanation for these results came from the experiments

performed by Stommel and Wahl (2004). They showed that phosphorylation of Mdm2 by DNA damage-induced PI3KKs (phospo-inositide-3-kinase-related protein kinase), including the S395 phosphorylation, temporarily destabilizes the Mdm2 protein (Stommel and Wahl, 2004). Incubation with a general inhibitor of PI3KK kinases, wortmannin, prevented the DSB-induced destabilization of Mdm2. Importantly, their experiments also indicated that destabilization of Mdm2 is essential for proper activation of p53.

It has recently been shown that ATM-mediated signaling also regulates the stability of Mdmx (Pereg et al., 2005). Mdmx was shown to be degraded in an Mdm2-dependent manner following DNA damage (Pan and Chen, 2003; de Graaf et al., 2003; Kawai et al., 2003a). The requirements for degradation of Mdmx and p53 by Mdm2 were observed to be different (de Graaf et al., 2003; Kawai et al., 2003b; Meulmeester et al., 2003). The central acidic domain of Mdm2 was found to be required for p53 ubiquitination and degradation of p53, while Mdmx ubiquitination requires only the Mdm2 RING domain (de Graaf et al., 2003; Meulmeester et al., 2003). Subsequently, ATM was found to phosphorylate Mdmx on Ser403 in response to DSB induction, and this phosphorylation contributes to DNA damage-induced ubiquitination and degradation of Mdmx. Accordingly, the degradation of Mdmx in ATM-null cells is retarded, although not completely abolished. Since blocking PI3KKs with wortmannin fully blocked DSB-induced degradation of Mdmx, other members of the PI3KK family might also be involved in this phosphorylation. Given the p53 paradigm, a search for additional phosphorylation sites in Mdmx identified two additional serines in Mdmx that showed increased phosphorylation upon DNA damage - Ser342 and Ser367. A putative dependence of Mdmx phosphorylation at Ser367 and Ser342 on the PI3KK kinases is under investigation. Significantly, mutating either Ser367 or Ser342 into alanine inhibited the damage-induced, Mdm2-dependent degradation of Mdmx, without affecting the interaction between Mdmx and Mdm2 (Pereg et al., 2005). In addition, Okamoto and coworkers showed that Ser367 and Ser342 phosphorylation creates high-affinity 14-3-3 binding sites (Okamoto and Jochemsen unpublished results). Indeed, serine to alanine mutation of Ser367 and Ser342 abolished or markedly reduced 14-3-3 binding,

respectively. As expected, the interaction between wildtype Mdmx and 14-3-3 proteins clearly increased upon DNA damage (K. Okamoto et al, submitted). This situation resembles the results observed with Raf, in which two binding sites in Raf cooperated to associate with 14-3-3 proteins (Tzivion et al., 1998). Interestingly, the S367A mutant did not enter the nucleus upon DNA damage, while the S342A mutant entered the nucleus less efficiently upon DNA damage compared to wild type Mdmx (A. Teunisse and A.G.Jochemsen, unpublished observations). The re-localization of Mdmx into the nucleus appears to be critical for inhibition of p53-mediated transcription (Migliorini et al., 2002). Transfection studies revealed that Mdm2 translocates Mdmx into the nucleus, requiring the RING domain and NLS of Mdm2 (Migliorini et al., 2002). In addition, p53 was found to be able to recruit Mdmx into the nucleus, independently of Mdm2, and complex formation between p53 and Mdmx was required for this effect. However the translocation of Mdmx was also observed in p53/mdm2 null cells, suggesting that Mdmx recruitment into the nucleus is not only dependent on p53 and Mdm2. These results could indicate 14-3-3 proteins as important regulators of Mdmx localization, and might explain the Mdm2 and p53-independent re-localization after DNA damage. The observation that the mutants S342A and S367A resulted in increased stability of Mdmx upon DNA damage, suggests a role for 14-3-3 proteins in Mdmx stability (Pereg et al., 2005). It must be noted that the role of 14-3-3 proteins in Mdmx stability and localization is based on correlation. One way to investigate the role of 14-3-3 proteins in Hdmx localization could be to deplete this protein, however, RNAi-mediated knockdown of 14-3-3 proteins is technically challenging due to functional redundancy of several isoforms. In addition, the binding between Mdmx and 14-3-3 proteins is dependent on phosphorylation, which probably implicates that overexpression of 14-3-3 proteins is not expected to decrease the stability or alter the localization of the Mdmx protein under normal growth conditions.

All in all, these data strongly suggest that the ATM-mediated DNA damage response targets Mdmx in multiple ways, resembling the regulation of p53, to ensure a tight regulation. These results together indicate that DNA-damage-activated PI3KKs modulate the abundance of p53’s negative regulators Mdmx

and Mdm2 to ensure proper p53 activation and subsequent activation of p53-mediated damage response pathways.

The mechanism by which stability of Mdmx and Mdm2 is decreased after DNA damage remained unknown. In figure 3 a model for control of the stability of Mdm2 and Mdmx upon DNA damage is depicted. While it had been shown that deubiquitination by HAUSP has a profound effect on the stability of both Mdm2 and p53, new evidence supports a role for HAUSP in the regulation of Mdmx stability as well (Cummins and Vogelstein, 2004; Li et al., 2004; Li et al., 2002b; Meulmeester et al., 2005). The deubiquitination enzyme HAUSP was shown to directly bind and deubiquitinate Mdmx in vitro and in vivo. The expression of HAUSP is critical to maintain Mdmx protein levels under normal growth conditions. Although overexpression of HAUSP resulted in increased levels of Mdmx, increased HAUSP levels could not prevent the DNA damage-induced degradation of Mdmx. Similarly, HAUSP overexpression did not inhibit the temporary destabilization of Mdm2 upon DSB-induction. Even though the intrinsic enzymatic activity of HAUSP was not decreased after DNA damage, its ability to deubiquitinate Mdmx or Mdm2 was impaired. Interestingly the HAUSP-Mdmx and HAUSP-Mdm2 interactions were attenuated upon DNA damage, while the interaction between HAUSP and p53 was not diminished. Interestingly, and possibly expectedly in view of the observations mentioned above, the decreased HAUSP-Mdmx association upon DNA damage was dependent on phosphorylation of Mdmx, and could be largely restored by pre-treating the cells with the PI3KK-inhibitor caffeine. Based on the current data a model can be proposed in which ATM/ATR-mediated phosphorylation of Mdmx and Mdm2 results in a decreased interaction with HAUSP (figure 3). As a result, Mdmx and Mdm2 become highly unstable upon DNA damage and cannot inhibit p53 function anymore. On the other hand, ATM-mediated phosphorylation activates the p53 protein. Therefore, disruption of the HAUSP-Mdmx and HAUSP-Mdm2 interaction may prove to be a useful target for therapeutic intervention in the p53 pathway, to reactivate p53 in tumors with wild type p53.

Concluding remarks

In conclusion, since the identification of Mdmx in 1996, it has been established that Mdmx is a critical regulator of the p53 pathway. Initially, Mdmx was identified as a p53 interacting protein that could inhibit p53 mediated transcription. The embryonic lethality of mdmx knockout mice, which was rescued in a p53 null background, underscored the importance of this regulator in the p53 pathway. Subsequently, Mdmx was shown to comprise oncogene activity and was found overexpressed in various tumor cell lines and primary human tumors. Overexpression of Mdmx could thus be an important step for p53 inactivation in human tumor formation. Importantly, we have recently demonstrated that HAUSP mediated stabilization of Mdmx is attenuated upon DNA damage in an ATM dependent fashion, which contributes to the activation of the p53 pathway. Further understanding of the regulation of Mdmx may offer new tools for therapeutic intervention in the p53 pathway for cancer treatment.

References

Argentini,M., Barboule,N., and Wasylyk,B. (2001). The contribution of the acidic domain of MDM2 to p53 and MDM2 stability. Oncogene 20, 1267-1275.

Avantaggiati,M.L., Ogryzko,V., Gardner,K., Giordano,A., Levine,A.S., and Kelly,K. (1997). Recruitment of p300/CBP in p53-dependent signal pathways. Cell 89, 1175-1184.

Bakkenist,C.J. and Kastan,M.B. (2003). DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499-506.

Bakkenist,C.J. and Kastan,M.B. (2004). Initiating cellular stress responses. Cell 118, 9-17.

Banin,S., Moyal,L., Shieh,S., Taya,Y., Anderson,C.W., Chessa,L., Smorodinsky,N.I., Prives,C., Reiss,Y.,

Shiloh,Y., and Ziv,Y. (1998). Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281, 1674-1677.

Barak,Y., Juven,T., Haffner,R., and Oren,M. (1993). mdm2 expression is induced by wild type p53 activity. EMBO J.

12, 461-468.

Baskaran,R., Wood,L.D., Whitaker,L.L., Canman,C.E., Morgan,S.E., Xu,Y., Barlow,C., Baltimore,D., Wynshaw-Boris,A., Kastan,M.B., and Wang,J.Y. (1997). Ataxia telangiectasia mutant protein activates c-Abl tyrosine kinase in response to ionizing radiation. Nature 387, 516-519.

Bassing,C.H. and Alt,F.W. (2004). The cellular response to general and programmed DNA double strand breaks. DNA Repair (Amst) 3, 781-796.

Bottger,V., Bottger,A., Garcia-Echeverria,C., Ramos,Y.F., van der Eb,A.J., Jochemsen,A.G., and Lane,D.P. (1999). Comparative study of the p53-mdm2 and p53-MDMX interfaces. Oncogene 18, 189-199.

Boyd,S.D., Tsai,K.Y., and Jacks,T. (2000). An intact HDM2 RING-finger domain is required for nuclear exclusion of p53. Nat. Cell Biol. 2, 563-568.

Brignone,C., Bradley,K.E., Kisselev,A.F., and Grossman,S.R. (2004). A post-ubiquitination role for MDM2 and hHR23A in the p53 degradation pathway. Oncogene 23, 4121-4129.

Chehab,N.H., Malikzay,A., Appel,M., and Halazonetis,T.D. (2000). Chk2/hCds1 functions as a DNA damage

checkpoint in G(1) by stabilizing p53. Genes Dev. 14, 278-288.

Chuikov,S., Kurash,J.K., Wilson,J.R., Xiao,B., Justin,N., Ivanov,G.S., McKinney,K., Tempst,P., Prives,C., Gamblin,S.J., Barlev,N.A., and Reinberg,D. (2004). Regulation of p53 activity through lysine methylation. Nature 432, 353-360.

Cummins,J.M. and Vogelstein,B. (2004). HAUSP is Required for p53 Destabilization. Cell Cycle 3, 689-692.

D'Orazi,G., Cecchinelli,B., Bruno,T., Manni,I., Higashimoto,Y., Saito,S., Gostissa,M., Coen,S., Marchetti,A., Del Sal,G., Piaggio,G., Fanciulli,M.,

Appella,E., and Soddu,S. (2002). Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis. Nat. Cell Biol. 4, 11-19.

Danovi,D., Meulmeester,E., Pasini,D., Migliorini,D., Capra,M., Frenk,R., de Graaf,P., Francoz,S., Gasparini,P., Gobbi,A., Helin,K., Pelicci,P.G., Jochemsen,A.G., and Marine,J.C. (2004). Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity. Mol. Cell Biol. 24, 5835-5843.

de Graaf,P., Little,N.A., Ramos,Y.F., Meulmeester,E., Letteboer,S.J., and Jochemsen,A.G. (2003). Hdmx protein stability is regulated by the ubiquitin ligase activity of Mdm2. J. Biol. Chem. 278, 38315-38324.

Demonacos,C., Krstic-Demonacos,M., Smith,L., Xu,D., O'Connor,D.P., Jansson,M., and La Thangue,N.B. (2004). A new effector pathway links ATM kinase with the DNA damage response. Nat. Cell Biol. 6, 968-976.

Dornan,D. and Hupp,T.R. (2001). Inhibition of p53-dependent transcription by BOX-I phospho-peptide mimetics that bind to p300. EMBO Rep. 2, 139-144.

Dornan,D., Wertz,I., Shimizu,H., Arnott,D., Frantz,G.D., Dowd,P., O'Rourke,K., Koeppen,H., and Dixit,V.M. (2004). The ubiquitin ligase COP1 is a critical negative regulator of p53. Nature 429, 86-92.

Dumaz,N. and Meek,D.W. (1999). Serine15

phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2. EMBO J. 18, 7002-7010.

Finch,R.A., Donoviel,D.B., Potter,D., Shi,M., Fan,A., Freed,D.D., Wang,C.Y., Zambrowicz,B.P., Ramirez-Solis,R., Sands,A.T., and Zhang,N. (2002). mdmx is a negative regulator of p53 activity in vivo. Cancer Res. 62, 3221-3225.

Finlan,L. and Hupp,T.R. (2004). The N-terminal interferon-binding domain (IBiD) homology domain of p300 binds to peptides with homology to the p53 transactivation domain. J. Biol. Chem. 279, 49395-49405.

Gatei,M., Sloper,K., Sorensen,C., Syljuasen,R., Falck,J., Hobson,K., Savage,K., Lukas,J., Zhou,B.B., Bartek,J., and Khanna,K.K. (2003). Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation. J. Biol. Chem. 278, 14806-14811.

Geyer,R.K., Yu,Z.K., and Maki,C.G. (2000). The MDM2 RING-finger domain is required to promote p53 nuclear export. Nat. Cell Biol. 2, 569-573.

Glockzin,S., Ogi,F.X., Hengstermann,A., Scheffner,M., and Blattner,C. (2003). Involvement of the DNA repair protein hHR23 in p53 degradation. Mol. Cell Biol. 23, 8960-8969.

Goldberg,Z., Vogt,S.R., Berger,M., Zwang,Y., Perets,R., Van Etten,R.A., Oren,M., Taya,Y., and Haupt,Y. (2002). Tyrosine phosphorylation of Mdm2 by c-Abl: implications for p53 regulation. EMBO J. 21, 3715-3727.

Gostissa,M., Hengstermann,A., Fogal,V., Sandy,P., Schwarz,S.E., Scheffner,M., and Del Sal,G. (1999). Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1. EMBO J. 18, 6462-6471.

Grossman,S.R., Deato,M.E., Brignone,C., Chan,H.M., Kung,A.L., Tagami,H., Nakatani,Y., and Livingston,D.M. (2003). Polyubiquitination of p53 by a ubiquitin ligase activity of p300. Science 300, 342-344.

Grossman,S.R., Perez,M., Kung,A.L., Joseph,M., Mansur,C., Xiao,Z.X., Kumar,S., Howley,P.M., and Livingston,D.M. (1998). p300/MDM2 complexes participate in MDM2-mediated p53 degradation. Mol. Cell

2, 405-415.

Gu,J., Kawai,H., Nie,L., Kitao,H., Wiederschain,D., Jochemsen,A.G., Parant,J., Lozano,G., and Yuan,Z.M. (2002). Mutual dependence of MDM2 and MDMX in their functional inactivation of p53. J. Biol. Chem. 277, 19251-19254.

Gu,W. and Roeder,R.G. (1997). Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595-606.

Haupt,Y., Maya,R., Kazaz,A., and Oren,M. (1997). Mdm2 promotes the rapid degradation of p53. Nature 387, 296-299.

Hollstein,M., Sidransky,D., Vogelstein,B., and Harris,C.C. (1991). p53 mutations in human cancers. Science 253, 49-53.

Honda,R., Tanaka,H., and Yasuda,H. (1997). Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 420, 25-27.

Issaeva,N., Bozko,P., Enge,M., Protopopova,M.,

Verhoef,L.G., Masucci,M., Pramanik,A., and Selivanova,G. (2004). Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors. Nat. Med. 10, 1321-1328.

Ito,A., Kawaguchi,Y., Lai,C.H., Kovacs,J.J.,

Higashimoto,Y., Appella,E., and Yao,T.P. (2002). MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation. EMBO J. 21, 6236-6245.

Jackson,M.W. and Berberich,S.J. (2000). MdmX protects p53 from Mdm2-mediated degradation. Mol. Cell Biol. 20, 1001-1007.

Jones,S.N., Ansari-Lari,M.A., Hancock,A.R., Jones,W.J., Gibbs,R.A., Donehower,L.A., and Bradley,A. (1996). Genomic organization of the mouse double minute 2 gene. Gene 175, 209-213.

Jones,S.N., Roe,A.E., Donehower,L.A., and Bradley,A. (1995). Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208.

Juven-Gershon,T. and Oren,M. (1999). Mdm2: the ups and downs. Mol. Med. 5, 71-83.

Kawai,H., Nie,L., Wiederschain,D., and Yuan,Z.M. (2001). Dual role of p300 in the regulation of p53 stability. J. Biol. Chem. 276, 45928-45932.

Kawai,H., Wiederschain,D., Kitao,H., Stuart,J., Tsai,K.K., and Yuan,Z.M. (2003a). DNA damage-induced MDMX degradation is mediated by MDM2. J. Biol. Chem. 278, 45946-45953.

Kawai,H., Wiederschain,D., and Yuan,Z.M. (2003b). Critical contribution of the MDM2 acidic domain to p53 ubiquitination. Mol. Cell Biol. 23, 4939-4947.

Kubbutat,M.H., Jones,S.N., and Vousden,K.H. (1997). Regulation of p53 stability by Mdm2. Nature 387, 299-303.

Kurz,E.U. and Lees-Miller,S.P. (2004). DNA damage-induced activation of ATM and ATM-dependent signaling pathways. DNA Repair (Amst) 3, 889-900.

Lai,Z., Ferry,K.V., Diamond,M.A., Wee,K.E., Kim,Y.B., Ma,J., Yang,T., Benfield,P.A., Copeland,R.A., and Auger,K.R. (2001). Human mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism requiring enzyme isomerization. J. Biol. Chem. 276, 31357-31367.

Lambert,P.F., Kashanchi,F., Radonovich,M.F.,

Shiekhattar,R., and Brady,J.N. (1998). Phosphorylation of p53 serine 15 increases interaction with CBP. J. Biol. Chem. 273, 33048-33053.

Leng,R.P., Lin,Y., Ma,W., Wu,H., Lemmers,B., Chung,S., Parant,J.M., Lozano,G., Hakem,R., and Benchimol,S. (2003). Pirh2, a p53-induced ubiquitin-protein ligase, promotes p53 degradation. Cell 112, 779-791.

Li,C., Chen,L., and Chen,J. (2002a). DNA damage induces MDMX nuclear translocation by p53dependent and -independent mechanisms. Mol. Cell Biol. 22, 7562-7571.

Li,M., Brooks,C.L., Kon,N., and Gu,W. (2004). A dynamic role of HAUSP in the p53-Mdm2 pathway. Mol. Cell 13, 879-886.

Li,M., Brooks,C.L., Wu-Baer,F., Chen,D., Baer,R., and Gu,W. (2003). Mono- versus polyubiquitination: differential control of p53 fate by Mdm2. Science 302, 1972-1975.

Li,M., Chen,D., Shiloh,A., Luo,J., Nikolaev,A.Y., Qin,J., and Gu,W. (2002b). Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature 416, 648-653.

Lill,N.L., Grossman,S.R., Ginsberg,D., DeCaprio,J., and Livingston,D.M. (1997). Binding and modulation of p53 by p300/CBP coactivators. Nature 387, 823-827.

Linares,L.K., Hengstermann,A., Ciechanover,A., Muller,S., and Scheffner,M. (2003). HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53. Proc. Natl. Acad. Sci. U. S. A 100, 12009-12014.

Lukas,J., Lukas,C., and Bartek,J. (2004). Mammalian cell cycle checkpoints: signalling pathways and their

organization in space and time. DNA Repair (Amst) 3, 997-1007.

Matsuoka,S., Huang,M., and Elledge,S.J. (1998). Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 282, 1893-1897.

Maya,R., Balass,M., Kim,S.T., Shkedy,D., Leal,J.F., Shifman,O., Moas,M., Buschmann,T., Ronai,Z., Shiloh,Y., Kastan,M.B., Katzir,E., and Oren,M. (2001). ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes Dev. 15, 1067-1077.

Meulmeester,E., Frenk,R., Stad,R., de Graaf,P.,

Marine,J.C., Vousden,K.H., and Jochemsen,A.G. (2003). Critical role for a central part of Mdm2 in the ubiquitylation of p53. Mol. Cell Biol. 23, 4929-4938.

Meulmeester,E., Maurice,M.M., Boutell,C., Teunisse,A.F., Ovaa,H., Abraham,T.E., Dirks,R.W., and Jochemsen,A.G. (2005). Loss of HAUSP-Mediated Deubiquitination Contributes to DNA Damage-Induced Destabilization of Hdmx and Hdm2. Mol. Cell 18, 565-576.

Michael,D. and Oren,M. (2002). The p53 and Mdm2 families in cancer. Curr. Opin. Genet. Dev. 12, 53-59.

Migliorini,D., lazzerini Denchi,E., Danovi,D.,

Jochemsen,A., Capillo,M., Gobbi,A., Helin,K., Pelicci,P.G., and Marine,J.C. (2002). Mdm4 (Mdmx) regulates

induced growth arrest and neuronal cell death during early embryonic mouse development. Mol. Cell Biol. 22, 5527-5538.

Minsky,N. and Oren,M. (2004). The RING domain of Mdm2 mediates histone ubiquitylation and transcriptional repression. Mol. Cell 16, 631-639.

Momand,J., Wu,H.H., and Dasgupta,G. (2000). MDM2--master regulator of the p53 tumor suppressor protein. Gene

242, 15-29.

Montes de Oca Luna,R., Wagner,D.S., and Lozano,G. (1995). Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206.

Pan,Y. and Chen,J. (2003). MDM2 promotes ubiquitination and degradation of MDMX. Mol. Cell Biol. 23, 5113-5121.

Parant,J., Chavez-Reyes,A., Little,N.A., Yan,W.,

Reinke,V., Jochemsen,A.G., and Lozano,G. (2001). Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53. Nat. Genet. 29, 92-95.

Pereg,Y., Shkedy,D., de Graaf,P., Meulmeester,E., Edelson-Averbukh,M., Salek,M., Biton,S., Teunisse,A.F., Lehmann,W.D., Jochemsen,A.G., and Shiloh,Y. (2005). Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage. Proc. Natl. Acad. Sci. U. S. A 102, 5056-5061.

Prives,C. (1998). Signaling to p53: breaking the MDM2-p53 circuit. Cell 95, 5-8.

Ramos,Y.F., Stad,R., Attema,J., Peltenburg,L.T., van der Eb,A.J., and Jochemsen,A.G. (2001). Aberrant expression of HDMX proteins in tumor cells correlates with wild-type p53. Cancer Res. 61, 1839-1842.

Riemenschneider,M.J., Buschges,R., Wolter,M., Reifenberger,J., Bostrom,J., Kraus,J.A., Schlegel,U., and Reifenberger,G. (1999). Amplification and overexpression of the MDM4 (MDMX) gene from 1q32 in a subset of malignant gliomas without TP53 mutation or MDM2 amplification. Cancer Res. 59, 6091-6096.

Riemenschneider,M.J., Knobbe,C.B., and Reifenberger,G. (2003). Refined mapping of 1q32 amplicons in malignant gliomas confirms MDM4 as the main amplification target. Int. J. Cancer 104, 752-757.

Rodriguez,M.S., Desterro,J.M., Lain,S., Midgley,C.A., Lane,D.P., and Hay,R.T. (1999). SUMO-1 modification activates the transcriptional response of p53. EMBO J. 18, 6455-6461.

Sabbatini,P. and McCormick,F. (2002). MDMX inhibits the p300/CBP-mediated acetylation of p53. DNA Cell Biol. 21, 519-525.

Saito,S., Goodarzi,A.A., Higashimoto,Y., Noda,Y., Lees-Miller,S.P., Appella,E., and Anderson,C.W. (2002). ATM mediates phosphorylation at multiple p53 sites, including

Ser(46), in response to ionizing radiation. J. Biol. Chem.

277, 12491-12494.

Saito,S., Yamaguchi,H., Higashimoto,Y., Chao,C., Xu,Y., Fornace,A.J., Jr., Appella,E., and Anderson,C.W. (2003). Phosphorylation site interdependence of human p53 post-translational modifications in response to stress. J. Biol. Chem. 278, 37536-37544.

Sakaguchi,K., Saito,S., Higashimoto,Y., Roy,S.,

Anderson,C.W., and Appella,E. (2000). Damage-mediated phosphorylation of human p53 threonine 18 through a cascade mediated by a casein 1-like kinase. Effect on Mdm2 binding. J. Biol. Chem. 275, 9278-9283.

Schon,O., Friedler,A., Bycroft,M., Freund,S.M., and Fersht,A.R. (2002). Molecular mechanism of the interaction between MDM2 and p53. J. Mol. Biol. 323, 491-501.

Scolnick,D.M., Chehab,N.H., Stavridi,E.S., Lien,M.C., Caruso,L., Moran,E., Berger,S.L., and Halazonetis,T.D. (1997). CREB-binding protein and p300/CBP-associated factor are transcriptional coactivators of the p53 tumor suppressor protein. Cancer Res. 57, 3693-3696.

Shafman,T., Khanna,K.K., Kedar,P., Spring,K., Kozlov,S., Yen,T., Hobson,K., Gatei,M., Zhang,N., Watters,D., Egerton,M., Shiloh,Y., Kharbanda,S., Kufe,D., and Lavin,M.F. (1997). Interaction between ATM protein and c-Abl in response to DNA damage. Nature 387, 520-523.

Sharp,D.A., Kratowicz,S.A., Sank,M.J., and George,D.L. (1999). Stabilization of the MDM2 oncoprotein by interaction with the structurally related MDMX protein. J. Biol. Chem. 274, 38189-38196.

Shieh,S.Y., Ahn,J., Tamai,K., Taya,Y., and Prives,C. (2000). The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev. 14, 289-300.

Shiloh,Y. (2003). ATM and related protein kinases: safeguarding genome integrity. Nat. Rev. Cancer 3, 155-168.

Shiloh,Y. and Lehmann,A.R. (2004). Maintaining integrity. Nat. Cell Biol. 6, 923-928.

Shinozaki,T., Nota,A., Taya,Y., and Okamoto,K. (2003). Functional role of Mdm2 phosphorylation by ATR in attenuation of p53 nuclear export. Oncogene 22, 8870-8880.

Shvarts,A., Steegenga,W.T., Riteco,N., van Laar,T., Dekker,P., Bazuine,M., van Ham,R.C., van der Houven van Oordt, Hateboer,G., van der Eb,A.J., and Jochemsen,A.G. (1996). MDMX: a novel p53-binding protein with some functional properties of MDM2. EMBO J. 15, 5349-5357.

Sledz,C.A., Holko,M., De Veer,M.J., Silverman,R.H., and Williams,B.R. (2003). Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5, 834-839.

Stad,R., Little,N.A., Xirodimas,D.P., Frenk,R., van der Eb,A.J., Lane,D.P., Saville,M.K., and Jochemsen,A.G.

(2001). Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms. EMBO Rep. 2, 1029-1034.

Stad,R., Ramos,Y.F., Little,N., Grivell,S., Attema,J., Der Eb,A.J., and Jochemsen,A.G. (2000). Hdmx stabilizes Mdm2 and p53. J. Biol. Chem. 275, 28039-28044.

Steinman,H.A., Sluss,H.K., Sands,A.T., Pihan,G., and Jones,S.N. (2004). Absence of p21 partially rescues Mdm4 loss and uncovers an antiproliferative effect of Mdm4 on cell growth. Oncogene 23, 303-306.

Stommel,J.M. and Wahl,G.M. (2004). Accelerated MDM2 auto-degradation induced by DNA-damage kinases is required for p53 activation. EMBO J. 23, 1547-1556.

Tanimura,S., Ohtsuka,S., Mitsui,K., Shirouzu,K., Yoshimura,A., and Ohtsubo,M. (1999). MDM2 interacts with MDMX through their RING finger domains. FEBS Lett. 447, 5-9.

Tzivion,G., Luo,Z., and Avruch,J. (1998). A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. Nature 394, 88-92.

Vassilev,L.T. (2004). Small-molecule antagonists of p53-MDM2 binding: research tools and potential therapeutics. Cell Cycle 3, 419-421.

Vogelstein,B., Lane,D., and Levine,A.J. (2000). Surfing the p53 network. Nature 408, 307-310.

Vousden,K.H. (2000). p53: death star. Cell 103, 691-694.

Waterman,M.J., Stavridi,E.S., Waterman,J.L., and

Halazonetis,T.D. (1998). ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins. Nat. Genet. 19, 175-178.

Xirodimas,D.P., Saville,M.K., Bourdon,J.C., Hay,R.T., and Lane,D.P. (2004). Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity. Cell 118, 83-97.

Xirodimas,D.P., Stephen,C.W., and Lane,D.P. (2001). Cocompartmentalization of p53 and Mdm2 is a major determinant for Mdm2-mediated degradation of p53. Exp. Cell Res. 270, 66-77.

Yuan,Z.M., Huang,Y., Ishiko,T., Nakada,S., Utsugisawa,T., Shioya,H., Utsugisawa,Y., Yokoyama,K.,

Weichselbaum,R., Shi,Y., and Kufe,D. (1999). Role for p300 in stabilization of p53 in the response to DNA damage. J. Biol. Chem. 274, 1883-1886.

Zacchi,P., Gostissa,M., Uchida,T., Salvagno,C., Avolio,F., Volinia,S., Ronai,Z., Blandino,G., Schneider,C., and Del Sal,G. (2002). The prolyl isomerase Pin1 reveals a mechanism to control p53 functions after genotoxic insults. Nature 419, 853-857.

Zheng,H., You,H., Zhou,X.Z., Murray,S.A., Uchida,T., Wulf,G., Gu,L., Tang,X., Lu,K.P., and Xiao,Z.X. (2002). The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response. Nature 419, 849-853.

Zhu,Q., Yao,J., Wani,G., Wani,M.A., and Wani,A.A. (2001). Mdm2 mutant defective in binding p300 promotes ubiquitination but not degradation of p53: evidence for the role of p300 in integrating ubiquitination and proteolysis. J. Biol. Chem. 276, 29695-29701.

Chapter 2

Critical Role for a Central Part of Mdm2 in the

Ubiquitylation of p53

Mol. Cell. Biol. (2003) 23:4929-4938

MOLECULAR AND CELLULAR BIOLOGY, July 2003, p. 4929–4938 Vol. 23, No. 14 0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.14.4929–4938.2003

Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Critical Role for a Central Part of Mdm2 in the Ubiquitylation of p53

Karen H. Vousden,3 _{and Aart G. Jochemsen}1_*

Department of Molecular and Cell Biology and Center for Biomedical Genetics, Leiden University Medical Center, 2300 RA Leiden, The Netherlands1; ULB-IBMM, Laboratoire d’Embryologie Moléculaire, 6041 Gosselies, Belgium2;

and Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, United Kingdom3

Received 4 December 2002/Returned for modification 22 January 2003/Accepted 9 April 2003

The stability of the p53 protein is regulated by Mdm2. By acting as an E3 ubiquitin ligase, Mdm2 directs the ubiquitylation of p53 and its subsequent degradation by the 26S proteasome. In contrast, the Mdmx protein, although structurally similar to Mdm2, cannot ubiquitylate or degrade p53 in vivo. To ascertain which domains determine this functional difference between Mdm2 and Mdmx and consequently are essential for p53 ubiquitylation and degradation, we generated Mdm2-Mdmx chimeric constructs. Here we show that, in addition to a fully functional Mdm2 RING finger, an internal domain of Mdm2 (residues 202 to 302) is essential for p53 ubiquitylation. Strikingly, the function of this domain can be fulfilled in trans, indicating that the RING domain and this internal region perform distinct activities in the ubiquitylation of p53.

The tumor suppressor protein p53 is a transcription factor that functions to inhibit cell proliferation and is therefore usually maintained at low levels in cells to allow normal growth. p53 is stabilized and activated in response to several forms of cellular stress, leading to the induction of a set of target genes involved in cell cycle arrest, DNA repair, or apoptosis, dependent on the type and strength of the signal (32). An important regulator of p53 activity is Mdm2, which inhibits the function of p53 both by abolishing its transcription-regulatory activity (4, 24) and by targeting p53 for degradation (10, 17). Mdm2 is an E3 ubiquitin ligase that directs the ubiquitylation of both p53 and Mdm2 (6, 11, 12), resulting in the degradation of both proteins by the 26S proteasome. Mdm2 is also a transcriptional target of p53 (21, 27), and so p53 and Mdm2 form a tight autoregulatory feedback loop, the importance of which has been genetically demonstrated by the ability of p53-null mice to rescue the embryonic lethality of mdm2-null mice (16, 25). The Mdmx protein is structurally homologous to Mdm2 (29). The highest conservation between Mdmx and Mdm2 is found in three regions named CR1, CR2, and CR3. CR1 (residues 42 to 94) contains the p53-binding domain, CR2 (residues 301 to 329) spans a putative Zn finger domain, and CR3 (residues 444 to 483) harbors the RING finger domain required for ubiquitin ligase activity. In contrast to Mdm2, Mdmx contains no nuclear localization signal (NLS) or nuclear export signal (NES), and the acidic region (residues 237 to 274) is less well conserved at the primary amino acid level between the two.

* Corresponding author. Mailing address: Leiden University Medical Center, Department of Molecular and Cell Biology, P.O. Box 9503, 2300 RA Leiden, The Netherlands. Phone: 31 715276136. Fax: 31 71 5276284. E-mail: A.G.Jochemsen@lumc.nl.

† Present address: University of Amsterdam, Faculty of Sciences, 1090 GB Amsterdam, The Netherlands.

proteins, although Mdmx also contains many acidic amino acids in the same region. Due to the lack of an NLS or NES, the subcellular localization of the Mdmx protein is determined by its association with other cellular proteins, providing a level of regulation of Mdmx activity. It has been shown by us and others that in several cell types Mdmx is mainly

cytoplasmic but is efficiently translocated into the nucleus by coexpression of Mdm2 and/or p53 (8, 18, 22). In addition, a p53- and Mdm2-independent nuclear translocation after induction of DNA damage has been shown (18). However, in some cell types we find a constitutive predominantly nuclear localization of Mdmx (e.g., in C33A cells) (31). In spite of its inability to ubiquitylate or degrade p53 in vivo, Mdmx can inhibit transcription activation by p53 (15, 20, 30). Importantly, mdmx-null mice show embryonic lethality, a phenotype that can be rescued by p53 deficiency, showing that—like Mdm2—Mdmx is a critical regulator of p53 in vivo (7, 23, 26).

C Considering the functional differences between Mdm2 and Mdmx, we generated a set of Mdm2-Mdmx hybrids to investigate the roles of different domains needed for p53 ubiquitylation and degradation. We show that, in addition to the RING finger, a central domain in Mdm2 is essential for p53 ubiquitylation.

MATERIALS AND METHODS

Plasmids. Expression vectors for p53, a p53 NLS mutant, Mdm2, an Mdm2

VOL.23,2003 CENTRAL PART OF Mdm2 ESSENTIAL FOR p53 UBIQUITYLATION 4930

FIG. 1. The p53-binding domains of Mdm2 and Mdmx are functionally similar. (A) Schematic representation of the Mdmx and Mdm2 proteins. Functional domains are indicated with the positions of the endonuclease recognition sites used to generate the chimeric constructs. Amino acid changes caused by the introduction of these sites are indicated. (B) p53- and mdm2-null cells (in 5-cm-diameter dishes) were transfected in duplicate with 200 ng of p53 without or with 1 µg of the indicated constructs in the presence of 1 µg of a His-tagged ubiquitin expression construct. Cells were either treated with 20 µM MG132 or mock treated 4 h before harvesting. The lysates from the MG132-treated cells were used to detect ubiquitylated p53 (His purified), while the lysates from the mock-treated cells were analyzed for total levels of p53. (C) C33A cells were transfected with the indicated constructs and analyzed by immunofluorescence 40 h after transfection. Mdm2 and 2xx were detected with the anti-Mdm2 antibody 4B2 (a and d), Mdmx was detected with the anti-Mdmx antibody 6B1A (g), and x22 was detected with the anti-Mdm2 antibody 2A10 (j). p53 was detected with the anti-p53 antibody FL393 (b, e, h, and k). Nuclei were stained with 4’,6’-diamidino-2-phenylindole (DAPI) (c, f, i, and l).

alterations and eventual amino acid sequence alterations are shown in Fig. 1A. All cDNAs were cloned into pcDNA3.1 (Invitrogen). All plasmids produced by PCR were checked by sequencing and restriction fragment analysis. The sequences of all primers used in the cloning or mutagenesis are available upon request.

Cell lines, cell culture, and transfection. H1299 cells were cultured in

RPMI medium with 10% fetal bovine serum (FBS). Approximately 8 h prior to transfection, the medium was changed to Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS. C33A cells were maintained in DMEM with 10% FBS. H1299 and C33A cells were transfected by the calcium phosphate coprecipitation method as described previously (31). p53- and mdm2-null cells were cultured in DMEM with 10% FBS and transfected with Fugene6 transfection reagent (Roche Molecular Diagnostics) according to the manufacturer’s protocol.

In vivo ubiquitylation assay. Forty hours after transfection, cells were

washed twice and scraped in ice-cold phosphate-buffered saline (PBS). Ten percent (10%) of the cell suspension was lysed in IPB 0.7 (20 mM triethanolamine [pH7.4], 0.7 M NaCl, 0.5% NP-40, 0.2% sodium deoxycholic acid) or in Giordano buffer (50 mM Tris [pH 7.4], 250 mM NaCl, 0.1% Triton X-100, 5 mM EDTA). Lysis of the remaining 90% of the cells, subsequent isolation of His-ubiquitinconjugated protein, and analysis of eluates and total lysates by Western blotting were performed as described previously (30).

Antibodies. The following antibodies were used: the anti-p53 mouse

VOL.23,2003 CENTRAL PART OF Mdm2 ESSENTIAL FOR p53 UBIQUITYLATION 4931

rabbit polyclonal antibody p57. p57 is a mouse Mdmx-specific rabbit serum raised against an internal peptide of Mdmx (amino acids [aa] 179 to 195). LacZ was detected with the mouse monoclonal antibody D19-2F3-2 (Roche Molecular Diagnostics).

Immunofluorescence. Forty hours after transfection, C33A cells were

washed twice with PBS, fixed for 15 min in 4% paraformaldehyde in PBS, and permeabilized for 10 min with 0.2% Triton X-100 in PBS at room temperature. Incubation of the cells with primary and secondary antibodies was performed as described previously (31).

RESULTS

In previous studies it was found that Hdmx appeared to be incapable of enhancing the ubiquitylation of p53 in vivo (30,31). To further investigate whether Hdmx would have any detectable ubiquitin ligase activity, two different in vitro Ubiquitylation assays were performed by use of UbcH5 as the E2 enzyme. In both an autoubiquitylation assay and a p53 ubiquitylation assay, Hdmx failed to display any significant activity, while all known E3 ligases tested (e.g., Mdm2 and Praja) showed self-ubiquitylation activity and Mdm2 could ubiquitylate p53. These results strongly suggest that the RING domain of Hdmx does not act as an E3 ubiquitin ligase, although the possibility that a different E2 enzyme is essential for Hdmx to function as such cannot be completely excluded. To study which domains determine this functional difference between Mdmx and Mdm2, we decided to generate Mdm2-Mdmx chimeric constructs.

Generation and nomenclature of the Mdm2-Mdmx chimeric constructs. To investigate in more detail the domains in Mdm2

necessary for in vivo ubiquitylation and degradation of p53, we made mouse Mdmx chimeras. Initially we divided the Mdm2-Mdmx proteins into three parts: the p53-binding domain (p53-BD; aa 1 to 106), the middle domain (aa 107 to 303), and the zinc finger/RING finger domain (ZF/RF; aa 304 to 489). These three regions are also the basis of our nomenclature for the different chimeras; e.g., x22 contains the p53-BD of Mdmx and the middle domain and ZF/RF of Mdm2 (Fig. 1A). Subsequently, the middle and ZF/RF domains were each divided into two parts, and the order of these exchanges is indicated in parentheses; e.g., 2(2-x)2 contains the p53-BD, the NLS/NES region, and the ZF/RF region of Mdm2 but the acidic domain-containing region (AD) of Mdmx. To construct these chimeras, endonucle se recognition sites were created in Mdmx and Mdm2 cDNAs on analogous sites, with minimal changes on the amino acid level (Fig. 1A). Amino acid changes in Mdmx were chosen such that the sequence would be more like Mdm2. The few amino acid changes that were introduced into Mdm2 did not affect the function of full-length Mdm2 (data not shown).

The p53-binding domains of Mdmx and Mdm2 are functionally similar. We and others have shown that the p53- binding domains of

Hdm2 and Hdmx bind p53 with similar requirements (3, 8). To investigate whether these domains can functionally replace each other regarding ubiquitylation and degradation of p53, they were exchanged between Mdmx and Mdm2 (Fig. 1A). These chimeras, x22 and 2xx, were coexpressed with p53 in p53- and mdm2-null cells, with wild-type Mdm2 and Mdmx as controls. Duplicate transfected dishes were either incubated with the proteasome inhibitor MG132 (to allow detection of p53 ubiquitylation) or mock treated (to allow detection of p53 degradation) 4 h prior to harvesting of the cells. The degradation and in vivo ubiquitylation of p53 were investigated as described previously (30, 33). The results show that the x22 hybrid can ubiquitylate and degrade p53 as efficiently as Mdm2 (Fig. 1B, lanes 2 and 5), while the 2xx hybrid, like Mdmx, lacks both activities (Fig. 1B, lanes 3 and 4). The Mdmx and 2xx

proteins are both cytoplasmic and nuclear in the p53- and mdm2-null cells. However, directing the expression of these proteins exclusively to the nucleus by fusing the simian virus 40 NLS at the N terminus does not change the inability to ubiquitylate or degrade p53 (data not shown). To confirm these activities in an independent assay, the same constructs were expressed in C33A cells in order to investigate their abilities to degrade endogenous mutant p53. Again, only Mdm2 (Fig. 1Ca, b, and c) and the x22 chimera (Fig. 1C j, k, and l) degraded p53. A protein is scored as being able to degrade p53 when at least 80% of strongly positive cells show a significant reduction in the p53 signal. We found with the in vivo ubiquitylation assay that the ubiquitylation of endogenous mutant p53 in C33A cells is increased upon transfection of Mdm2 or a degrading chimera, while a nondegrading Mdm2 mutant or chimera has no effect (data not shown). This result indicates that degradation of p53, as scored by immunofluorescence, and enhanced ubiquitylation are correlated. As mentioned above, in the C33A cells the Mdmx protein, as well as 2xx, is mainly nuclear. This localization is dependent on an intact Mdmx RING domain (see also below). Since these results show that the origin of the p53-binding domain does not affect the Ubiquitylation and degradation of p53, most subsequent chimeras tested contained the p53-binding domain of Mdm2, allowing simultaneous detection of the Mdm2–Mdmx chimeras with the anti-Mdm2 antibody 4B2.

A complete Mdm2 RING is essential for efficient ubiquitylation and degradation of p53. As a basis for further chimeras we

exchanged the ZF/RF domain of Mdm2 for that of Mdmx, creating 22x (Fig. 2A). This chimeric protein, as well as the derived RING finger chimeras (see below), contains the NLS/NES region of Mdm2 and is predominantly located in the nucleus. As expected, the 22x chimeric protein could not ubiquitylate or degrade p53 in p53- and

mdm2-null cells or in H1299 cells (Fig. 2B and C, lanes 5 and 6). In

4932 MEULMEESTER ET AL. MOL. CELL. BIOL.

FIG. 2. Mdm2-Mdmx swaps at the C terminus of the RING finger inhibit ubiquitin ligase activity. (A) Schematic representation of Mdmx-Mdm2 chimeras with exchanges at the C terminus of the RING domain. (B) p53- and mdm2-null cells were transfected in duplicate with 200 ng of p53, with or without 1 µg of the indicated constructs, and with 1 µg of the His-ubiquitin expression vector and 1 µg of CMV-LacZ. Cells were either treated with 20 µM MG132 or mock treated 4 h before harvesting. Western blot analysis was performed on ubiquitylated proteins (His-purified) and total cell lysates. p53 was detected with DO-1, Mdm2 and chimeric proteins were detected with 4B2, and LacZ was detected with D19-2F3-2. (C) H1299 cells (in 9.4-cm-diameter dishes) were transfected with 500 ng of p53, with or without 2 µg of constructs, and with 2 µg of a His-ubiquitin expression vector and 2 µg of CMV-LacZ. Transfected cells were treated, and lysates were subsequently analyzed, as described for panel B.

these chimeric proteins to degrade p53 was confirmed by transfection into C33A cells and investigation of the level of endogenous p53 by immunofluorescence (data not shown). To investigate whether a small N-terminal part of the Mdm2 RING could be functionally replaced by the analogous part of the Mdmx RING, we created chimeras by use of the AgeI site at aa 453 and 454, generating 22(2-x)A and 22(x-2)A. The 22(x-2)A construct was also made with the N447C mutation (Fig. 3A). These chimeras were coexpressed with

VOL.23,2003 CENTRAL PART OF Mdm2 ESSENTIAL FOR p53 UBIQUITYLATION 4933

FIG. 3. Mdm2-Mdmx exchanges at the N terminus of the RING finger prevent complete p53 ubiquitylation. (A) Schematic representation of the Mdmx-Mdm2 chimeras with exchanges at the N terminus of the RING or containing the complete Mdm2 RING. (B and C) p53- and mdm2- null cells (B) and H1299 cells (C) were transfected in duplicate with the indicated constructs; ubiquitylated proteins and total lysates were analyzed as described for Fig. 2B. (D) Immunofluorescence analysis of C33A cells transfected with 22(x-2)A, 22(x-2)A N447C, or 22(x-2)Afl. Cells were stained for expression of the chimeric proteins with 4B2 (a, d, and g) and for expression of p53 with FL393 (b, e, and h). Nuclei were stained with 4’,6’-diamidino-2-phenylindole (DAPI) (c, f, and i).

lack of degradation can be explained by the fact that the high-molecularweight ubiquitylated p53 proteins were underrepresented relative to the low-molecular-weight ubiquitylated p53 species, compared to the ubiquitylation pattern of p53 observed after coexpression with Mdm2. In addition, relative to the total level of p53, the level of ubiquitylated species is much lower than that seen after coexpression of Mdm2. Taken together, these results indicate that a complete Mdm2 RING is necessary for efficient polyubiquitylation of p53 such that it can be degraded by the