MDM2 promotes ubiquitination and degradation of MDMX - PubMed (original) (raw)

MDM2 promotes ubiquitination and degradation of MDMX

Yu Pan et al. Mol Cell Biol. 2003 Aug.

Abstract

The p53 tumor suppressor is regulated by MDM2-mediated ubiquitination and degradation. Mitogenic signals activate p53 by induction of ARF expression, which inhibits p53 ubiquitination by MDM2. Recent studies showed that the MDM2 homolog MDMX is also an important regulator of p53. We present evidence that MDM2 promotes MDMX ubiquitination and degradation by the proteasomes. This effect is stimulated by ARF and correlates with the ability of ARF to bind MDM2. Promotion of MDM2-mediated MDMX ubiquitination requires the N-terminal domain of ARF, which normally inhibits MDM2 ubiquitination of p53. An intact RING domain of MDM2 is also required, both to interact with MDMX and to provide E3 ligase function. Increase of MDM2 and ARF levels by DNA damage, recombinant ARF adenovirus infection, or inducible MDM2 expression leads to proteasome-mediated down-regulation of MDMX levels. Therefore, MDMX and MDM2 are coordinately regulated by stress signals. The ARF tumor suppressor differentially regulates the ability of MDM2 to promote p53 and MDMX ubiquitination and activates p53 by targeting both members of the MDM2 family.

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Figures

FIG. 1.

MDMX ubiquitination and degradation by MDM2. (A) MDMX was coexpressed with His6-ubiquitin in H1299 cells by transient transfection. His6-ubiquitinated proteins were purified, and MDMX was detected by Western blotting with 8C6 antibody (top panel). The lower panels are control Western blots of nonpurified cell lysate (10% of the amount used for Ni-NTA purification). (B) H1299 cells transfected with the indicated plasmids were treated with 30 μM MG132 for 4 h prior to Western blot analysis for MDMX. Transfection efficiency was verified by expression of cotransfected GFP plasmid. (C) MDM2/p53-null 174.1 cells were transfected with the indicated plasmids. Ni-NTA-purified ubiquitinated MDMX and cell lysate were run on the same gel to show the molecular weight shift due to MDM2-independent monoubiquitination.

FIG. 2.

MDM2 domains required for promoting MDMX ubiquitination. (A) H1299 cells were transfected with His6-ubiquitin, MDMX, and representative MDM2 mutants. MDMX ubiquitination and degradation were detected by Ni-NTA purification and Western blotting. (B) H1299 cells were transfected with His6-ubiquitin, MDM2, and MDMX mutants. MDMX ubiquitination was detected by Ni-NTA purification and Western blotting. (C) Diagram of MDM2 and MDMX mutants and summary of results. MDMX-ΔN has a deletion of residues 1 to 100. MDMX-ΔC has a deletion of residues 394 to 490.

FIG. 3.

Ubiquitination of MDMX requires MDM2 E3 function. (A) Position of point mutations in the MDM2 RING domain. C and H residues important for zinc coordination are underlined. Ubiquitination of MDMX by MDM2 mutants was determined by transfection of H1299 cells as described in the legend for Fig. 1A. (B) Binding of MDMX to MDM2 point mutants. MDMX and MDM2 mutants were cotransfected into H1299 cells and analyzed by MDM2 immunoprecipitation followed by MDMX Western blotting.

FIG. 4.

The ARF N-terminal domain is sufficient to cooperate with MDM2. (A) H1299 cells were transfected with MDMX, a suboptimal amount of MDM2, and different ARF mutants. His6-ubiquitin-conjugated MDMX was purified and detected by Western blotting. (B) Diagram of ARF mutants and summary of results.

FIG. 5.

DNA damage and ARF expression down-regulate MDMX level. (A) U2OS cells were infected with ARF adenovirus at the indicated multiplicity for 24 h and analyzed for MDMX, MDM2, and p53 expression levels by direct Western blotting. (B) Primary HFFs were treated with 10 Gy of gamma radiation and analyzed for MDMX, MDM2, p53, and ARF levels by Western blotting at the indicated times after treatment. (C) Gamma irradiation does not reduce the MDMX mRNA level. Cells were treated with gamma radiation, and 6 h later total RNA was isolated and hybridized with an MDMX cDNA probe in a Northern blotting analysis.

FIG. 6.

Induction of MDM2 results in MDMX degradation by proteasomes. (A) HFFs and H1299 cells were treated with 10 Gy of gamma radiation for 6 h. MG132 (30 μM) was added immediately after radiation. The MDMX level was determined by Western blotting. (B) U2OS cells stably expressing tetracycline-repressible MDM2 were cultured in the presence or absence of 0.5 μg of tetracycline/ml for 20 h. MG132 was added for the last 4 h. MDMX and MDM2 levels were determined by Western blotting.

FIG. 7.

Inhibition of MDMX degradation by C-terminal epitope tags. (A) H1299 cells were transfected with MDMX with different epitope tags at the N or C terminus and subjected to Ni-NTA purification and Western blotting. The MDMX-myc-His6 protein was completely recovered by Ni-NTA beads as expected. (B) Diagrams of epitope-tagged MDMX and summary of results. C-terminal epitope tags block polyubiquitination and degradation by MDM2.

FIG. 8.

MDM2 promotes MDMX polyubiquitination in vitro. GST-MDM2, GST-RING (containing MDM2 residues 290 to 491), and GST-N (containing MDM2 residues 1 to 150) were purified using glutathione agarose beads. Loaded beads were incubated with in vitro-translated MDMX in the presence or absence of E1 and E2 in a ubiquitination reaction as described in Materials and Methods. Polyubiquitinated MDMX appears as a high-molecular-weight smear above the unmodified MDMX band.

FIG. 9.

MDMX is regulated by stress signals. MDM2 and MDMX cooperate to inhibit p53 activity in the absence of stress. Stress signals activate p53 by targeting both MDM2 and MDMX. Oncogenes such as E2F1 and c-myc induce ARF expression, which activates p53 by inhibiting p53 ubiquitination and promoting MDMX ubiquitination by MDM2. DNA damage activates p53 and induces MDM2 expression, which in turn degrades MDMX and leads to further activation of p53. DNA damage also promotes MDMX nuclear translocation, which may expose it to degradation by MDM2 but also enables it to more efficiently inhibit p53 at certain stages of the damage response.

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MDM2 promotes ubiquitination and degradation of MDMX - PubMed (original) (raw)