Inhibition of p53 DNA Binding Function by the MDM2 Protein Acidic Domain

Cross, Brittany; Chen, Lihong; Qian, Cheng; Li, Baozong; Yuan, Zhi-Min; Chen, Jiandong

doi:10.1074/jbc.m111.228981

Cited by 60 publications

(59 citation statements)

References 63 publications

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“…Phosphorylation of the acidic domain by GSK3 stimulates ubiquitination of p53 (22,23). We showed that the acidic domain also causes conformational changes in the p53 core and that the MDM2-p53 complex does not bind DNA (10). The MDM2 acidic domain-p53 core interaction may serve to position the C-terminal RING for efficient ubiquitination of p53 C-terminal lysines or expose the target lysines in the p53 core domain.…”

Section: Discussionmentioning

confidence: 92%

“…Our results showed that the structural integrity of this region is critical for p53 ubiquitination. We recently also showed that ARF inhibits the ability of MDM2 acidic domain to induce p53 misfolding (10). Therefore, ATM phosphorylation of MDM2 and ARF binding appear to exert similar effects on the acidic domain function (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…We recently found that the MDM2 acidic domain is responsible for the misfolding of p53 (10), providing a functional assay to examine the effect of C-terminal phosphorylation on the acidic domain. In the p53 conformation assay, cotransfection of MDM2 with wild-type p53 caused a switch of p53 antibody reactivity from Pab1620 positive (wild type specific) to Pab240 positive (mutant specific) if p53 degradation is blocked by MG132 (10,36). We found that IR treatment prevented the conformational switch induced by MDM2 (Fig.…”

Section: Mdm2 Ring Domain Interaction and Regulation In Vivomentioning

confidence: 99%

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Regulation of MDM2 E3 Ligase Activity by Phosphorylation after DNA Damage

Qian

Cross

et al. 2011

Molecular and Cellular Biology

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MDM2 is a major regulator of p53 by acting as a ubiquitin E3 ligase. The central acidic domain and C-terminal RING domain of MDM2 are both indispensable for ubiquitination of p53. Our previous study suggested that ATM phosphorylation of MDM2 near the C terminus inhibits RING domain oligomerization, resulting in p53 stabilization after DNA damage. We present here evidence that these modifications allosterically regulate the functions of both acidic domain and RING domain of MDM2. Using chemical cross-linking, we show that the MDM2 RING domain forms oligomers including dimer and higher-order complexes in vivo. RING domain dimerization efficiency is negatively regulated by upstream sequence. ATM-mediated phosphorylation of the upstream sequence further inhibits RING dimerization. Forced oligomerization of MDM2 partially overcomes the inhibitory effect of phosphorylation and stimulates p53 ubiquitination. Furthermore, the ability of MDM2 acidic domain to bind p53 core domain and induce p53 misfolding are also suppressed by the same C-terminal ATM sites after DNA damage. These results suggest that the acidic domain and RING domain of MDM2 are both allosterically coupled to the intervening ATM sites, which enables the same modification to regulate multiple MDM2 functions critical for p53 ubiquitination.The p53 pathway is critical for maintenance of genomic stability and preventing development of cancer. The most notable feature of p53 is its stabilization after exposure to a wide range of stress signals such as hyperproliferation, nucleotide depletion, and DNA damage. These responses may be essential for its function as a tumor suppressor (14). In normal cells, p53 is present at very low levels due to rapid degradation through the ubiquitin-dependent proteasome pathway. p53 turnover is regulated by MDM2, which binds p53 and functions as an ubiquitin E3 ligase to promote p53 degradation by the proteasome (15,16,21). Additional E3 ligases such as Pirh2 and Cop1 have also been implicated as regulators of p53 turn over (12,24). However, their physiological significance in p53 regulation and stress response still remain to be established. Current evidence suggests that MDM2 is a major and indispensable regulator of p53 level (18,32).MDM2 and p53 interact through their N-terminal domains in a high-affinity binding and through their central domains in a weak binding (48). Upon complex formation, the MDM2 C-terminal RING domain recruits ubiquitin-conjugating enzyme E2 that performs the transfer of activated ubiquitin to p53 lysine residues. To date, the understanding of molecular mechanisms that lead to p53 stabilization by different pathways remain incomplete. An important group of MDM2 regulators are proteins that bind to the central acidic domain, such as ARF, L5, L11, and L23 (37, 50). These basic proteins are important for mediating mitogenic stress and ribosomal stress signals to p53. They have been shown to inhibit MDM2-mediated p53 ubiquitination, but little is known about the mechanisms. They may act by neutralizing ...

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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Mdm2 Ring Domain Interaction and Regulation In Vivomentioning

confidence: 99%

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Regulation of MDM2 E3 Ligase Activity by Phosphorylation after DNA Damage

Qian

Cross

et al. 2011

Molecular and Cellular Biology

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“…(27)(28)(29), the deubiquitinating enzyme HAUSP (30), ribosomal proteins (19), and the tumor suppressor ARF (31). Furthermore, the MDM2 acidic domain can bind weakly to the p53 core domain and induces p53 conformational change (32)(33)(34)(35)(36). The flexibility of the acidic domain is probably critical for interactions with multiple protein partners (37,38).…”

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confidence: 99%

Autoactivation of the MDM2 E3 Ligase by Intramolecular Interaction

Qian

Song

Chen

2014

Molecular and Cellular Biology

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The RING domain ubiquitin E3 ligase MDM2 is a key regulator of p53 degradation and a mediator of signals that stabilize p53. The current understanding of the mechanisms by which MDM2 posttranslational modifications and protein binding cause p53 stabilization remains incomplete. Here we present evidence that the MDM2 central acidic region is critical for activating RING domain E3 ligase activity. A 30-amino-acid minimal region of the acidic domain binds to the RING domain through intramolecular interactions and stimulates the catalytic function of the RING domain in promoting ubiquitin release from charged E2. The minimal activation sequence is also the binding site for the ARF tumor suppressor, which inhibits ubiquitination of p53. The acidic domain-RING domain intramolecular interaction is modulated by ATM-mediated phosphorylation near the RING domain or by binding of ARF. These results suggest that MDM2 phosphorylation and association with protein regulators share a mechanism in inhibiting the E3 ligase function and stabilizing p53 and suggest that targeting the MDM2 autoactivation mechanism may be useful for therapeutic modulation of p53 levels.A unique feature of the p53 tumor suppressor is its stabilization after exposure to many stress signals. This leads to the induction of numerous transcriptional targets that inhibit cell cycle progression, induce apoptosis, and regulate energy metabolism (1). The MDM2 and MDMX proteins are responsible for establishing the dynamic features of the p53 pathway. MDM2 is a RING domain ubiquitin (Ub) E3 ligase for p53 that promotes p53 degradation (2, 3). Mouse models provided strong evidence that MDM2 is indispensable for controlling p53 activity at all stages of life (4-6). The stabilization of p53 by small-molecule inhibitors that disrupt p53-MDM2 binding also confirmed that MDM2 is a major regulator of p53 turnover (7,8). MDM2-p53 disruptors have antitumor activity in animal models, and their potential as cancer drugs is currently being tested in the clinic (9).Numerous stress signals have been shown to cause p53 accumulation, mainly by inhibiting its degradation. MDM2 promotes p53 degradation by forming a stable complex through N-terminal domains. The MDM2 C-terminal RING domain recruits ubiquitin-conjugating enzyme E2, which performs a covalent modification of p53 lysine residues (10, 11). The major E2 isoforms involved in MDM2-mediated p53 ubiquitination in cells belong to the UbcH5 family (12). The MDM2-UbcH5 combination promotes the synthesis of mainly K48-linked polyubiquitin chains on p53 that target p53 for degradation by the 26S proteasome. MDM2-mediated ubiquitination of p53 is inhibited by multiple mechanisms. Phosphorylation of the p53 N terminus after DNA damage reduces MDM2 binding and contributes to p53 activation (13,14). DNA damage also induces ATM-dependent phosphorylation of MDM2, which inhibits RING domain dimerization and p53 polyubiquitination (15-17). Oncogene activation induces the expression of ARF, which binds to MDM2 and inhibits p53 ubiquitina...

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“…Recent results show that bortezomib induces the binding of p53 on DNA, promotes transcription factors and inhibits MDM2 [15].…”

Section: Inhibition Of P53mentioning

confidence: 99%

Bortezomib in Anti-Cancer Activity: A Potential Drug

Appavu

Deepa²

2016

Glob J Cancer Ther

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Abstract26S proteasome is an intracellular; ATP dependent enzymatic complex degrades ubiquitin-tagged proteins and maintains cellular homeostasis. The orderly degraded proteins including cyclins, caspases, Bcl-xL, p53, cell adhesion molecules are involved in cell-cycle progression, tumor suppression, DNA replication, inflammation, and apoptosis. So, proteasome inhibition is a target therapy for cancer to promote cell cycle arrest or apoptosis. Bortezomib (Velcade Proteasome inhibitorsA variety of natural and synthetic proteasome inhibitors are available such as peptide aldehydes, peptide α-ketoaldehydes, peptide vinyl sulfones, lactacystin, epoxomicin, peptide macrocycles, γ-lactam thiol ester, epipolythio-dioxopiperazine toxin and eponemycin. But many of these inhibitors are lack in specificity, poor metabolic stability, fast dissociation rate, irreversible and low affinity of binding makes them less suitable for use. For example, peptide aldehyde inhibitors dissociate rapidly and inactivated by oxidization which allows only short-lived proteasome inhibition. These problems overcome by replacing the corresponding aldehyde group in peptides by boronic acid [2]. Because it is forming stable tetrahedral transition state intermediate with N-terminal threonine residue of β5 active site and also peptide boranates dissociate more slowly and attaining stable inhibition [7].

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Inhibition of p53 DNA Binding Function by the MDM2 Protein Acidic Domain

Cited by 60 publications

References 63 publications

Regulation of MDM2 E3 Ligase Activity by Phosphorylation after DNA Damage

Regulation of MDM2 E3 Ligase Activity by Phosphorylation after DNA Damage

Autoactivation of the MDM2 E3 Ligase by Intramolecular Interaction

Bortezomib in Anti-Cancer Activity: A Potential Drug

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