Autoactivation of the MDM2 E3 Ligase by Intramolecular Interaction

Qian, Cheng; Song, Tanjing; Chen, Jiandong

doi:10.1128/mcb.00246-14

Cited by 20 publications

(32 citation statements)

References 62 publications

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“…Our analysis also detected a previously unidentified AD-RING internal binding in MDMX. We recently showed that the AD-RING internal interaction in MDM2 activates the ubiquitin E3 ligase activity of the RING domain (20). Here we found that in MDMX, the AD-RING binding is involved in suppressing nuclear translocation, presumably by concealing the NLS in the RING domain (16).…”

Section: Discussionmentioning

confidence: 61%

Autoinhibition of MDMX by intramolecular p53 mimicry

Chen¹,

Borcherds

Wu³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The p53 inhibitor MDMX is controlled by multiple stress signaling pathways. Using a proteolytic fragment release (PFR) assay, we detected an intramolecular interaction in MDMX that mechanistically mimics the interaction with p53, resulting in autoinhibition of MDMX. This mimicry is mediated by a hydrophobic peptide located in a long disordered central segment of MDMX that has sequence similarity to the p53 transactivation domain. NMR spectroscopy was used to show this hydrophobic peptide interacts with the N-terminal domain of MDMX in a structurally analogous manner to p53. Mutation of two critical tryptophan residues in the hydrophobic peptide disrupted the intramolecular interaction and increased p53 binding, providing further evidence for mechanistic mimicry. The PFR assay also revealed a second intramolecular interaction between the RING domain and central region that regulates MDMX nuclear import. These results establish the importance of intramolecular interactions in MDMX regulation, and validate a new assay for the study of intramolecular interactions in multidomain proteins with intrinsically disordered regions.T he p53 tumor suppressor is activated by numerous cellular and environmental signals and induces the expression of genes that regulate metabolism, cell growth, cell cycle, apoptosis, and senescence (1). MDM2 and MDMX are key regulators that control the cellular level and transcriptional activity of p53 through direct binding. Mouse knockout experiments showed that both MDM2 and MDMX are essential for controlling p53 activity during embryogenesis. Somatic knockout experiments showed that MDM2 is indispensable for regulating p53 in adult tissues, whereas MDMX deletion does not lead to cell death (2). MDM2 is a well-established p53 transcriptional target that forms a negative feedback loop by binding to the N-terminal transcriptional activation domain of p53 and, subsequently, ubiquitinating the C-terminal regulatory domain, which leads to degradation of p53 by the proteasome. p53 binding sites are also found in intron 1 of human MDMX, and p53 activation leads to moderate induction of MDMX transcription (3). Therefore, MDMX is a p53 target gene that may also provide dynamic feedback in response to p53 activation.MDMX alone does not have E3 ligase activity, but it is important for regulating p53 transcriptional function. MDMX expression and phosphorylation by the ATM/Chk2 pathway is important for the p53-mediated DNA damage response in mice (4, 5). MDMX levels are controlled by MDM2-mediated ubiquitination in a stress-dependent fashion (6, 7). Significant degradation of MDMX occurs after DNA damage through phosphorylation at several C-terminal sites (S342 and S367 by Chk2, S403 by ATM) (8). Furthermore, ribosomal stress promotes MDMX degradation through L11-MDM2 interaction (9), and oncogenic stress promotes MDMX degradation through ARF expression (10). Therefore, key signaling mechanisms that block p53 degradation simultaneously enhance MDMX degradation by MDM2. These observations underscore the co...

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Section: Discussionmentioning

confidence: 61%

Autoinhibition of MDMX by intramolecular p53 mimicry

Chen¹,

Borcherds

Wu³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…Recently, Cheng et al shown that the HDM2 RING domain is activated by intramolecular interaction with its acidic domain (40); thus, we hypothesized that the acidic domain in the construct HDM2(1-301) is able to recognize the RING domain of HDMX, since both RING domains have sequences with greater than 75% similarity (41) (Fig. 4g).…”

Section: Resultsmentioning

confidence: 96%

“…The E2-E3 pair could play a role in substrate specificity under different cellular conditions. Another question that arises from this work is the role of intramolecular interactions that has recently been described for HDMX (45,46) and for HDM2 (40). These intramolecular interactions could add another level of regulation to the way the domains are exposed to the solvent to allow highly accurate control of the interactome of HDM2.…”

Section: Discussionmentioning

confidence: 96%

Allosteric Interactions by p53 mRNA Govern HDM2 E3 Ubiquitin Ligase Specificity under Different Conditions

Medina-Medina

García-Beltrán

Mora

et al. 2016

Molecular and Cellular Biology

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c HDM2 and HDMX are key negative regulatory factors of the p53 tumor suppressor under normal conditions by promoting its degradation or preventing its trans activity, respectively. It has more recently been shown that both proteins can also act as positive regulators of p53 after DNA damage. This involves phosphorylation by ATM on serine residues HDM2(S395) and HDMX(S403), promoting their respective interaction with the p53 mRNA. However, the underlying molecular mechanisms of how these phosphorylation events switch HDM2 and HDMX from negative to positive regulators of p53 is not known. Our results show that these phosphorylation events reside within intrinsically disordered domains and change the conformation of the proteins. The modifications promote the exposition of N-terminal interfaces that support the formation of a new HDMX-HDM2 heterodimer independent of the C-terminal RING-RING interaction. The E3 ubiquitin ligase activity of this complex toward p53 is prevented by the p53 mRNA ligand but, interestingly, does not affect the capacity to ubiquitinate HDMX and HDM2. These results show how ATM-mediated modifications of HDMX and HDM2 switch HDM2 E3 ubiquitin ligase activity away from p53 but toward HDMX and itself and illustrate how the substrate specificity of HDM2 E3 ligase activity is regulated.T he activity of the RING-E3 ubiquitin ligase HDM2 is tightly regulated by posttranslational modifications and protein-protein interactions that control its subcellular localization and interacting partners (1, 2). The best-characterized activity of HMD2 is the N-terminal interaction between its hydrophobic pocket and the conserved BOX-I domain of p53, which under normal cellular conditions promotes p53 polyubiquitination. The nonredundant HDM2 paralogue HDMX (also called HDM4) is, like HDM2, upregulated in human cancers (3)(4)(5). Despite the similarity with the C-terminal RING domain of HDM2 (approximately 75%), HDMX does not harbor E3 ubiquitin ligase activity toward p53, and its negative activity is instead linked to suppression of p53 trans activity. Mice lacking either mdm2 (the hdm2 mouse orthologous) or mdmx (the hdmx mouse orthologous) die early during development in a p53-dependent manner (6-8). It has been shown more recently that HDMX assists HDM2-mediated degradation of p53 by stabilizing HDM2 via a C-terminal RING-RING interaction and also by promoting HMD2-mediated polyubiquitination of p53 in the cytoplasm (9, 10).The ataxia telangiectasia mutated (ATM) kinase is a key regulator of p53 activity during the double-stranded DNA damage response that helps to induce p53 expression and activity. The direct and indirect phosphorylation of residues within the p53 BOX-I domain of p53 prevents the interaction with HDM2 and is linked to an increase in p53 activity (11-13). The phosphorylation on HDM2 at serine 395 (394 in mouse MDM2) is crucial for p53 stabilization, and animals carrying the S394A mutation have an impaired response to irradiation (14). The corresponding mutation in the human protein prevents ...

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“…3F). Biologically, the acidic domain is a core domain for the intramolecular binding between the acidic and RING domains, which also mediates the interaction with HAUSP (19, 20). Because the KQKQ-MDM2 mutant bound HAUSP more strongly than did the wild-type MDM2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Acetylation-dependent regulation of MDM2 E3 ligase activity dictates its oncogenic function

et al. 2017

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Abnormal activation of the oncogenic E3 ubiquitin ligase murine double minute 2 (MDM2) is frequently observed in human cancers. By ubiquitinating the tumor suppressor p53 protein, which leads to its proteasome-mediated destruction, MDM2 limits the tumor-suppressing activity of p53. On the other hand, by ubiquitinating itself, MDM2 targets itself for destruction and promotes the p53 tumor suppressor pathway, a process that can be antagonized by the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP). We investigated the regulation of MDM2 substrate specificity and found that acetyltransferase p300–mediated acetylation and stabilization of MDM2 are molecular switches that block self-ubiquitination, thereby shifting its E3 ligase activity toward p53. In vitro and in cancer cell lines, p300-mediated acetylation of MDM2 on Lys182 and Lys185 enabled HAUSP to bind, presumably deubiquitinate, and stabilize MDM2. This acetylation within the nuclear localization signal domain decreased its interaction with the acidic domain, subsequently increased the interaction between the acidic domain and RING domain in MDM2, enabled the binding of HAUSP to the acidic domain in MDM2, and shifted MDM2 activity from autoubiquitination to p53 ubiquitination. However, upon genotoxic stress through exposure to etoposide, the deacetylase sirtuin 1 (SIRT1) deacetylated MDM2 at Lys182 and Lys185, thereby promoting self-ubiquitination and less ubiquitination and subsequent degradation of p53, thus increasing p53-dependent apoptosis. Therefore, this study indicates that dynamic acetylation is a molecular switch in the regulation of MDM2 substrate specificity, revealing further insight into the posttranslational regulation of the MDM2/p53 cell survival axis.

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Autoactivation of the MDM2 E3 Ligase by Intramolecular Interaction

Cited by 20 publications

References 62 publications

Autoinhibition of MDMX by intramolecular p53 mimicry

Autoinhibition of MDMX by intramolecular p53 mimicry

Allosteric Interactions by p53 mRNA Govern HDM2 E3 Ubiquitin Ligase Specificity under Different Conditions

Acetylation-dependent regulation of MDM2 E3 ligase activity dictates its oncogenic function

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