MDMX is essential for the regulation of p53 protein levels in the absence of a functional MDM2 C-terminal tail

Abstract Background MDM2 is an E3 ubiquitin ligase that is able to ubiquitinate p53, targeting it for proteasomal degradation. Its homologue MDMX does not have innate E3 activity, but is able to dimerize with MDM2. Although mouse models have demonstrated both MDM2 and MDMX are individually essential for p53 regulation, the significance of MDM2-MDMX heterodimerization is only partially understood and sometimes controversial. MDM2C462A mice, where the C462A mutation abolishes MDM2 E3 ligase activity as well as its ability to dimerize with MDMX, die during embryogenesis. In contrast, the MDM2Y487A mice, where the Y487A mutation at MDM2 C-terminus significantly reduces its E3 ligase activity without disrupting MDM2-MDMX binding, survive normally even though p53 is expressed to high levels. This indicates that the MDM2-MDMX heterodimerization plays a critical role in the regulation of p53. However, it remains unclear whether MDMX is essential for the regulation of p53 protein levels in the context of an endogenous MDM2 C-terminal tail mutation. Results Here, we studied the significance of MDM2-MDMX binding in an MDM2 E3 ligase deficient context using the MDM2Y487A mouse embryonic fibroblast (MEF) cells. Surprisingly, down-regulation of MDMX in MDM2Y487A MEFs resulted in a significant increase of p53 protein levels. Conversely, ectopic overexpression of MDMX reduced p53 protein levels in MDM2Y487A MEFs. Mutations of the RING domain of MDMX prevented MDMX-MDM2 binding, and ablated MDMX-mediated suppression of p53 protein expression. Additionally, DNA damage treatment and nuclear sequestration of MDMX inhibited MDMX activity to suppress p53 protein expression. Conclusions These results suggest that MDMX plays a key role in suppressing p53 protein expression in the absence of normal MDM2 E3 ligase activity. We found that the ability of MDMX to suppress p53 levels requires MDM2 binding and its cytoplasmic localization, and this ability is abrogated by DNA damage. Hence, MDMX is essential for the regulation of p53 protein levels in the context of an MDM2 C-terminal mutation that disrupts its E3 ligase activity but not MDMX binding. Our study is the first to examine the role of MDMX in the regulation of p53 in the context of endogenous MDM2 C-terminal mutant MEF cells.

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Critical role of presenilin-dependent γ-secretase activity in DNA damage-induced promyelocytic leukemia protein expression and apoptosis

Cell Death and Differentiation ◽

10.1038/cdd.2012.162 ◽

2013 ◽

Vol 20 (4) ◽

pp. 639-648 ◽

Cited By ~ 6

Author(s):

H Song ◽

J Hyun Boo ◽

K Ho Kim ◽

C Kim ◽

Y-E Kim ◽

...

Keyword(s):

Dna Damage ◽

Protein Expression ◽

Critical Role ◽

Promyelocytic Leukemia ◽

Promyelocytic Leukemia Protein ◽

Secretase Activity

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Prognostic role of p53 protein expression in epidermoid carcinoma of the anal canal

International Journal of Radiation Oncology*Biology*Physics ◽

10.1016/s0360-3016(99)00188-1 ◽

1999 ◽

Vol 45 (2) ◽

pp. 309-314 ◽

Cited By ~ 30

Author(s):

C.Shun Wong ◽

Ming S Tsao ◽

Vinay Sharma ◽

William B Chapman ◽

Melania Pintilie ◽

...

Keyword(s):

Protein Expression ◽

Anal Canal ◽

P53 Protein ◽

Epidermoid Carcinoma ◽

Prognostic Role ◽

P53 Protein Expression

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Grape seed extract ameliorated Ehrlich solid tumor-induced hepatic tissue and DNA damage with reduction of PCNA and P53 protein expression in mice

Environmental Science and Pollution Research ◽

10.1007/s11356-021-13904-8 ◽

2021 ◽

Author(s):

Mabrouk Attia Abd Eldaim ◽

Ehab Tousson ◽

Mohamed Mohamed Soliman ◽

Ibrahim El Tantawy El Sayed ◽

Abdel Aleem H. Abdel Aleem ◽

...

Keyword(s):

Dna Damage ◽

Protein Expression ◽

Solid Tumor ◽

P53 Protein ◽

Grape Seed Extract ◽

Grape Seed ◽

Seed Extract ◽

Hepatic Tissue ◽

P53 Protein Expression

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Regulation of p53 protein expression in human breast cancer cell lines

Journal of Cell Science ◽

10.1242/jcs.105.3.607 ◽

1993 ◽

Vol 105 (3) ◽

pp. 607-612 ◽

Cited By ~ 1

Author(s):

B. Vojtesek ◽

D.P. Lane

Keyword(s):

Protein Expression ◽

Cell Lines ◽

P53 Protein ◽

Critical Role ◽

P53 Gene ◽

Breast Cancer Cell Lines ◽

Breast Cancers ◽

Human Breast ◽

Wild Type ◽

P53 Protein Expression

Mutation of the p53 gene is a common occurrence in human breast cancers but is by no means universal. However, even in tumours where the gene is not mutated altered levels of p53 protein are often detected. This is also observed in cell lines derived from human breast cancers. By transfecting such cell lines containing either wild type or mutant p53 genes with a temperature-sensitive mutant mouse p53 gene we have established that the cellular environment plays a critical role in the regulation of p53 protein expression. The results suggest that tumours that aberrantly express wild-type p53 may have lost the normal growth regulatory response to the protein and thus be functionally similar to those expressing the mutant protein.

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The Basally Expressed p53-Mediated Homeostatic Function

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.775312 ◽

2021 ◽

Vol 9 ◽

Author(s):

Isha Nagpal ◽

Zhi-Min Yuan

Keyword(s):

Animal Studies ◽

Close Association ◽

P53 Protein ◽

Critical Role ◽

Protein Levels ◽

Elevated Expression ◽

Homeostatic Function ◽

E6 Protein ◽

Insight Into

Apart from mutations in the p53 gene, p53 functions can be alternatively compromised by a decrease in nuclear p53 protein levels or activities. In accordance, enhanced p53 protein turnover due to elevated expression of the critical p53 E3 ligase MDM2 or MDM2/MDMX is found in many human cancers. Likewise, the HPV viral E6 protein-mediated p53 degradation critically contributes to the tumorigenesis of cervical cancer. In addition, growth-promoting signaling-induced cell proliferation is accompanied by p53 downregulation. Animal studies have also shown that loss of p53 is essential for oncogenes to drive malignant transformation. The close association between p53 downregulation and carcinogenesis implicates a critical role of basally expressed p53. In accordance, available evidence indicates that a reduced level of basal p53 is usually associated with disruption of homeostasis, suggesting a homeostatic function mediated by basal p53. However, basally expressed p53 under non-stress conditions is maintained at a relatively low abundance with little transcriptional activity, raising the question of how basal p53 could protect homeostasis. In this review, we summarize the findings pertinent to basal p53-mediated activities in the hope of developing a model in which basally expressed p53 functions as a barrier to anabolic metabolism to preserve homeostasis. Future investigation is necessary to characterize basal p53 functionally and to obtain an improved understanding of p53 homeostatic function, which would offer novel insight into the role of p53 in tumor suppression.

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EBV-miR-BHRF1-1 Targets p53 Gene: Potential Role in Epstein-Barr Virus Associated Chronic Lymphocytic Leukemia

Cancer Research and Treatment ◽

10.4143/crt.2019.457 ◽

2020 ◽

Vol 52 (2) ◽

pp. 492-504 ◽

Cited By ~ 1

Author(s):

Dan-Min Xu ◽

Yi-Lin Kong ◽

Li Wang ◽

Hua-Yuan Zhu ◽

Jia-Zhu Wu ◽

...

Keyword(s):

Protein Expression ◽

Epstein Barr Virus ◽

P53 Protein ◽

P53 Gene ◽

Lymphocytic Leukemia ◽

Barr Virus ◽

Cycle Arrest ◽

P53 Protein Expression ◽

Epstein Barr

PurposeThe purpose of this study was to investigate the prognostic impact of Epstein-Barr virus (EBV)–microRNA (miRNA, miR)-BHRF1-1 with chronic lymphocytic leukemia (CLL) as well as role of EBV-miR-BHRF1-1 in p53 gene.Materials and MethodsQuantitative reverse transcription–polymerase chain reaction and western blotting were used to quantify EBV-miR-BHRF1-1 and p53 expression in cultured CLL.Resultsp53 aberration was associated with the higher expression level of EBV-miR-BHRF1-1 (p < 0.001) which was also an independent prognostic marker for overall survival (p=0.028; hazard ratio, 5.335; 95% confidence interval, 1.193 to 23.846) in 97 newly-diagnosed CLL patients after adjusted with International Prognostic Index for patients with CLL. We identified EBV-miR-BHRF1-1 as a viral miRNA regulator of p53. EBV-miR-BHRF1-1 repressed luciferase reporter activity by specific interaction with the seed region within the p53 3′- untranslated region. Discordance of p53 messenger RNA and protein expression was associated with high EBV-miR-BHRF1-1 levels in CLL patients and cell lines. EBV-miR-BHRF1- 1 inhibition upregulated p53 protein expression, induced cell cycle arrest and apoptosis and decreased cell proliferation in cell lines. EBV-miR-BHRF1-1 mimics downregulated p53 protein expression, decreased cell cycle arrest and apoptosis, and induced cell proliferation in cell lines.ConclusionThis study supported the role of EBV-miR-BHRF1-1 in p53 regulation in vitro. Our results support the potential of EBV-miR-BHRF1-1 as a therapeutic target in EBV-associated CLL with p53 gene aberration.

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Ubiquitin–proteasome degradation of serum- and glucocorticoid-regulated kinase-1 (SGK-1) is mediated by the chaperone-dependent E3 ligase CHIP

Biochemical Journal ◽

10.1042/bj20060905 ◽

2006 ◽

Vol 400 (2) ◽

pp. 235-244 ◽

Cited By ~ 30

Author(s):

Larissa Belova ◽

Sanjay Sharma ◽

Deanna R. Brickley ◽

Jeremy R. Nicolarsen ◽

Cam Patterson ◽

...

Keyword(s):

Insulin Signalling ◽

Critical Role ◽

E3 Ligase ◽

Proteasomal Degradation ◽

Interacting Protein ◽

Protein Levels ◽

C Terminus ◽

Ubiquitin Proteasome ◽

Steady State Levels ◽

Phosphoinositide 3 Kinase

SGK-1 (serum- and glucocorticoid-regulated kinase-1) is a stress-induced serine/threonine kinase that is phosphorylated and activated downstream of PI3K (phosphoinositide 3-kinase). SGK-1 plays a critical role in insulin signalling, cation transport and cell survival. SGK-1 mRNA expression is transiently induced following cellular stress, and SGK-1 protein levels are tightly regulated by rapid proteasomal degradation. In the present study we report that SGK-1 forms a complex with the stress-associated E3 ligase CHIP [C-terminus of Hsc (heat-shock cognate protein) 70-interacting protein]; CHIP is required for both the ubiquitin modification and rapid proteasomal degradation of SGK-1. We also show that CHIP co-localizes with SGK-1 at or near the endoplasmic reticulum. CHIP-mediated regulation of SGK-1 steady-state levels alters SGK-1 kinase activity. These data suggest a model that integrates CHIP function with regulation of the PI3K/SGK-1 pathway in the stress response.

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APOBEC3B Induction Following DNA Damage Response Modulates the Survival and Treatment Response in Human Multiple Myeloma

Blood ◽

10.1182/blood-2019-130297 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4381-4381

Author(s):

Lijie Xing ◽

Jiye Liu ◽

Yuyin Li ◽

Liang Lin ◽

Kenneth Wen ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Damage ◽

Protein Expression ◽

Treatment Response ◽

Cell Lines ◽

Critical Role ◽

Dependent Manner ◽

Dna Damages ◽

Drug Responses

Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B, A3B) is one of 7-membered DNA cytosine deaminase family, causing cytosine-to-uracil (C-to-U) deamination in single-stranded DNA and promoting mutations in multiple human cancers including multiple myeloma (MM). High APOBEC3B expression is found in a significant portion of MM patients with MAF overexpression among t(14;16) and t(14;20). A3B upregulation is further associated with poor prognosis in MM, suggesting its role in the MM pathophysiology. However, approximately 23% MM patients with high APOBEC3 activity are associated with MAF/MAFA/MAFB translocations, the remainder of patients with high APOBEC3 carry neither translocations nor overexpression of these genes. Besides, studies are lacking on how A3B is regulated and the role of A3B in drug responses in MM. We here defined new mechanisms controlling A3B expression and further characterized its impact on treatment responses to current anti-MM therapies. Using qRT-PCR, A3B transcript is significantly higher than other members of the APOBEC3 gene family in MM cell lines (n=19) and MM patients, indicating that A3B may play a major role in MM. Using immunoblotting analysis, A3B protein expression was further confirmed in MM cell lines with various levels (n=10). Importantly, A3B mRNA upregulation by 1.34-42.64 folds was observed in CD138-purified cells from majorities of MM patients (83.3%) when compared to PBMC from the same individual (n=12). In MM cell lines without MAF/MAFA/MAFB translocation as a study model, higher A3B protein expression is associated with higher DNA damage levels as evidenced by higher γ-H2AX. These results suggest that A3B expression might be influenced by DNA damage levels in MM cells. Following a short time treatment of gamma-irradiation to cause DNA damages, A3B expression in viable MM cells was enhanced in a dose-dependent manner. We next treated MM cells (n=5) with common anti-MM drugs such as Melphalan (Mel) and Bortizomib (btz), both of which induce DNA damages, followed by examination of changes in A3B and γ-H2AX. Under sublethal treatment conditions of Mel or btz, A3B was consistently induced at both mRNA and protein levels in multiple MM cell lines regardless of the baseline A3B expression. Significantly, A3B was upregulated and associated with increased γ-H2AX in patient MM cells treated with Mel or btz under sub-lethal doses. Since DNA damages activate the ATR/ATM pathway, we next investigated whether these kinases mediate A3B induction following treatments with these compounds in MM cells. The presence of ATM or ATR inhibitors blocked A3B upregulated by these DNA damage-inducing treatments in MM cell lines (n=3), indicating an ATM/ATR-dependent pathway for A3B changes. Next, gene-specific CRISPR knock out (KO) and inducible-shRNA knockdown (KD) were used to determine the functional impact of perturbation of A3B in proliferation and survival of MM cells. Both KO and KD of A3B decreased growth and viability of MM cell lines regardless of sensitive or resistant to dexamethasone or lenalidomide. Using LIVE/DEAD fixable Aqua Stain and annexin V-based flow cytometric analysis, A3B inhibition enhanced growth arrest followed by apoptosis in MM cells. Significantly, A3B KD by its shRNA in RPMI8226 MM cells enhanced sensitivity to pomalidomide. Taken together, these data indicate that increased A3B level plays a critical role in MM cell survival and drug responses. DNA damages triggered by IR, Mel, or btz further enhance A3B expression via ATM/ATR pathway, which in turn increases subclonal diversity leading to drug resistance. The role of A3B in disease pathophysiology and progression, coupled with its function in mediating treatment response, suggest potential utility of targeting A3B in MM. Disclosures Munshi: Celgene: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Takeda: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

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