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The MDM2 RING domain and central acidic domain play distinct roles in MDM2 protein homodimerization and MDM2-MDMX protein heterodimerization.

ABSTRACT: The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. Intriguingly, deletion of a portion of the MDM2 central acidic domain selectively inhibits interaction with MDM2 while leaving intact the ability of MDM2 to interact with MDMX and to ubiquitinate p53. Further analysis of an MDM2 C-terminal deletion mutant reveals that the C-terminal residues of MDM2 are required for both MDM2 and MDMX interaction. Collectively, our results suggest a model in which MDM2-MDMX heterodimerization requires the extreme C terminus and proper RING domain structure of MDM2, whereas MDM2 homodimerization requires the extreme C terminus and the central acidic domain of MDM2, suggesting that MDM2 homo- and heterodimers utilize distinct MDM2 domains. Our study is the first to report mutations capable of separating MDM2 homo- and heterodimerization.

SUBMITTER: Leslie PL

PROVIDER: S-EPMC4432308 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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The MDM2 RING domain and central acidic domain play distinct roles in MDM2 protein homodimerization and MDM2-MDMX protein heterodimerization.

Leslie Patrick L PL Ke Hengming H Zhang Yanping Y

The Journal of biological chemistry 20150325 20

The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo ...[more]

PMID: 25809483

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Project description:IntroductionMany human breast cancers overexpress the E3 ubiquitin ligase MDM2 and its homolog MDMX. Expression of MDM2 and MDMX occurs in estrogen receptor ?-positive (ER?+) breast cancer and triple-negative breast cancer (TNBC). There are p53-independent influences of MDM2 and MDMX, and 80% of TNBC express mutant p53 (mtp53). MDM2 drives TNBC circulating tumor cells (CTCs) in mice, but the context-dependent influences of MDM2 and MDMX on different subtypes of breast cancers expressing mtp53 have not been determined.MethodsTo assess the context-dependent roles, we carried out MDM2 and MDMX knockdown in orthotopic tumors of TNBC MDA-MB-231 cells expressing mtp53 R280K and MDM2 knockdown in ER?+ T47D cells expressing mtp53 L194F. The corresponding cell proliferation was scored in vitro by growth curves and in vivo by orthotopic tumor volumes. Cell migration was assessed in vitro by wound-healing assays and cell intravasation in vivo by sorting GFP-positive CTCs by flow cytometry. The metastasis gene targets were determined by an RT-PCR array card screen and verified by qRT-PCR and Western blot analysis.ResultsKnocking down MDMX or MDM2 in MDA-MB-231 cells reduced cell migration and CTC detection, but only MDMX knockdown reduced tumor volumes at early time points. This is the first report of MDMX overexpression in TNBC enhancing the CTC phenotype with correlated upregulation of CXCR4. Experiments were carried out to compare MDM2-knockdown outcomes in nonmetastatic ER?+ T47D cells. The knockdown of MDM2 in ER?+ T47D orthotopic tumors decreased primary tumor volumes, supporting our previous finding that estrogen-activated MDM2 increases cell proliferation.ConclusionsThis is the first report showing that the expression of MDM2 in ER?+ breast cancer and TNBC can result in different tumor-promoting outcomes. Both MDMX and MDM2 overexpression in TNBC MDA-MB-231 cells enhanced the CTC phenotype. These data indicate that both MDM2 and MDMX can promote TNBC metastasis and that it is important to consider the context-dependent roles of MDM2 family members in different subtypes of breast cancer.

Dataset Information

The MDM2 RING domain and central acidic domain play distinct roles in MDM2 protein homodimerization and MDM2-MDMX protein heterodimerization.

Publications

The MDM2 RING domain and central acidic domain play distinct roles in MDM2 protein homodimerization and MDM2-MDMX protein heterodimerization.

Similar Datasets

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