Abnormal MDMX degradation in tumor cells due to ARF deficiency

Li, X; Gilkes, Daniele M.; Li, B; Qian, Cheng; Pernazza, Daniele; Lawrence, Harshani R.; Lawrence, Nicole; Chen, J

doi:10.1038/onc.2011.534

Cited by 26 publications

(27 citation statements)

References 52 publications

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“…Although studies have extensively demonstrated p53-dependent properties of Nutlin, p53-independent effects of Nutlin have also emerged. One observed p53-independent effect of Nutlin is the increase in Mdm2 protein levels (6,21), which our data indicate is due to Mdm2 protein stabilization. We had previously shown Mdm2 overexpression inhibits DNA break repair and promotes genome instability independent of p53 (10,11).…”

Section: Discussionsupporting

confidence: 48%

“…It was previously reported that cell lines treated with Nutlin can lead to increased levels of Mdm2 as detected by Western blot, and this can occur in the absence of p53 (6,8). To determine whether Nutlin influences Mdm2 protein stability, we evaluated Mdm2 protein levels following addition of the protein synthesis inhibitor, cycloheximide.…”

Section: Resultsmentioning

confidence: 95%

“…It has been reported that a consequence of Nutlin treatment is elevated Mdm2 protein levels, and this occurs in cells with or without p53 (6,8). While Mdm2 negatively regulates p53, Mdm2 also has p53-independent functions that negatively impact genome stability (9).…”

Section: Introductionmentioning

confidence: 99%

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Pharmacologically Increasing Mdm2 Inhibits DNA Repair and Cooperates with Genotoxic Agents to Kill p53-Inactivated Ovarian Cancer Cells

Carrillo

Hicks

Khabele

et al. 2015

Molecular Cancer Research

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The Mdm2 oncogene is a negative regulator of the p53 tumor suppressor and recently identified inhibitor of DNA break repair. Nutlin-3 is a small molecule inhibitor of Mdm2/p53 interaction that can induce apoptosis in cancer cells through activation of p53. While this is promising therapy for those cancers with wild-type p53, half of all human cancers have inactivated p53. Here, we reveal a previously unappreciated effect of Nutlin is inhibition of DNA break repair, stemming from its ability to increase Mdm2 protein levels. The Nutlin-induced increase in Mdm2 inhibited DNA double-strand break (DSB) repair and prolonged DNA damage response signaling independent of p53. Mechanistically, this effect of Nutlin required Mdm2 and acted through Nbs1 of the Mre11/Rad50/Nbs1 DNA repair complex. In ovarian cancer cells where >90% have inactivated p53, Nutlin combined with the genotoxic agents, cisplatin or etoposide, had a cooperative lethal effect resulting in increased DNA damage and apoptosis. Therefore, these data demonstrate an unexpected consequence of pharmacologically increasing Mdm2 levels that when utilized in combination with genotoxic agents induces synthetic lethality in ovarian cancer cells, and likely other malignant cell types, that have inactivated p53. Implications Data reveal a therapeutically beneficial effect of pharmacologically increasing Mdm2 levels combined with chemotherapeutic agents for malignancies that have lost functional p53.

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Section: Discussionsupporting

confidence: 48%

Section: Resultsmentioning

confidence: 95%

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Pharmacologically Increasing Mdm2 Inhibits DNA Repair and Cooperates with Genotoxic Agents to Kill p53-Inactivated Ovarian Cancer Cells

Carrillo

Hicks

Khabele

et al. 2015

Molecular Cancer Research

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“…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.…”

mentioning

confidence: 99%

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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“…Significant degradation of MDMX occurs after DNA damage through phosphorylation on several C-terminal sites (S342 and S367 by Chk2 and S403 by ATM), with S367 phosphorylation being the most critical (5,40). Furthermore, ribosomal stress promotes MDMX degradation through L11-MDM2 interaction (12), and oncogenic stress promotes MDMX degradation through ARF expression (26). Therefore, key signaling mechanisms that block p53 degradation also simultaneously enhance MDMX degradation by MDM2.…”

mentioning

confidence: 99%

Casein Kinase 1α Regulates an MDMX Intramolecular Interaction To Stimulate p53 Binding

Chen

Becker

et al. 2012

Molecular and Cellular Biology

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MDMX is an important regulator of p53 during embryonic development and malignant transformation. Previous studies showed that casein kinase 1␣ (CK1␣) stably associates with MDMX, stimulates MDMX-p53 binding, and cooperates with MDMX to inactivate p53. However, the mechanism by which CK1␣ stimulates MDMX-p53 interaction remains unknown. Here, we present evidence that p53 binding by the MDMX N-terminal domain is inhibited by the central acidic region through an intramolecular interaction that competes for the p53 binding pocket. CK1␣ binding to the MDMX central domain and phosphorylation of S289 disrupts the intramolecular interaction, allowing the N terminus to bind p53 with increased affinity. After DNA damage, the MDMX-CK1␣ complex is disrupted by Chk2-mediated phosphorylation of MDMX at S367, leading to reduced MDMX-p53 binding. Therefore, CK1␣ is an important functional partner of MDMX. DNA damage activates p53 in part by disrupting CK1␣-MDMX interaction and reducing MDMX-p53 binding affinity. The p53 tumor suppressor can be activated by numerous cellular and environmental signals and induces the expression of genes that regulate metabolism, cell growth, division, and apoptosis (49). MDM2 and MDMX are key regulatory proteins that control p53 level and transcriptional activity. Recent studies suggested that the MDM2/MDMX module is largely responsible for enabling p53 to be highly responsive to stress, thus maintaining genomic stability and enhancing cellular and organismal robustness. The importance of p53 in maintaining normal homeostasis is underscored by the frequent p53 mutation or MDM2/MDMX dysregulation in human cancer.MDM2 and MDMX are homologs that bind to p53 with high affinity (31). Genetic analyses showed that both MDM2 and MDMX are essential for controlling p53 activity during embryogenesis (17,35,38). The role of MDMX appears to be less critical than that of MDM2 in adult tissues, as shown by somatic knockout experiments (13,30,55). MDM2 is a classic p53 transcriptional target, forming a negative feedback loop (54). Bona fide p53 binding sites are also found in intron 1 of the human MDMX gene, and p53 activation leads to moderate induction of MDMX transcription (23, 42). Induction of MDMX by p53 was also observed in a mouse model after somatic deletion of casein kinase 1␣ (CK1␣), which triggers oncogenic stress and p53 activation (9). The magnitude of MDMX mRNA induction after p53 activation (1.5 to 3-fold) is moderate compared to that of MDM2 (Ͼ10-fold) and displays cell type specificity. Therefore, MDMX is a p53 target gene that may also provide a negative feedback response to p53 activation.Investigations of p53 activation by DNA damage suggest that phosphorylation of p53, MDM2, and MDMX cooperate to achieve rapid and robust responses. MDM2-p53 binding is essential for degradation of p53 and has been extensively studied. DNA double-strand breaks induce phosphorylation of p53 S15 by DNA-dependent protein kinase (DNA-PK) and ATM (1,33,45). ATM also activates Chk2, which in turn phosphorylates p53...

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Abnormal MDMX degradation in tumor cells due to ARF deficiency

Cited by 26 publications

References 52 publications

Pharmacologically Increasing Mdm2 Inhibits DNA Repair and Cooperates with Genotoxic Agents to Kill p53-Inactivated Ovarian Cancer Cells

Pharmacologically Increasing Mdm2 Inhibits DNA Repair and Cooperates with Genotoxic Agents to Kill p53-Inactivated Ovarian Cancer Cells

Autoinhibition of MDMX by intramolecular p53 mimicry

Casein Kinase 1α Regulates an MDMX Intramolecular Interaction To Stimulate p53 Binding

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