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

Abstract

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.

Fig. 1. p300 acetylates MDM2 largely at the Lys¹⁸² and Lys¹⁸⁵ residues within the NLS domain

(A) Western blotting (IB) after immunoprecipitation (IP) of endogenous MDM2 from U2OS whole-cell lysate (WCL). Immunoglobulin G (IgG) immunoprecipitation served as a negative control. Blots are representative of n = 2 biological replicates. (B) Western blotting analysis after immunoprecipitation of endogenous MDM2 from T47D or ZR75 whole-cell lysate. n = 2 biological replicates. Ac-K, Ac-K182. (C) Western blotting analysis after immunoprecipitation with hemagglutinin (HA) antibody from whole-cell lysate from 293T cells transfected with HA-MDM2 and Myc-p300, Flag-GCN5, Flag-PCAF, or Flag-TIP60α and then treated with 5 mM nicotinamide (NIC) and 1 μM trichostatin A (TSA). n = 2 biological replicates. EV, empty vector. (D) Sequence alignment of the putative acetylation sites of MDM2 in various species. (E) Schematic diagram of the functional domains of human MDM2 protein. (F and G) Western blotting analysis after immunoprecipitation with HA antibody (F) or MDM2 antibody (G) from whole-cell lysate from 293 cells transfected with HA-MDM2 constructs and/or Myc-p300 and treated with 5 mM NIC and 1 μM TSA. n = 2 or 3 biological replicates. WT, wild-type. (H) Same as in (F) using 293T cells. n = 2 biological replicates.

Fig. 2. SIRT1 interacts with and deacetylates MDM2 both in cells and in vitro

(A) Western blotting after immunoprecipitation with HA antibody in whole-cell lysate from 293T cells cotransfected with HA-MDM2 and the indicated Flag-tagged SIRT construct. n = 2 biological replicates. (B and C) Schematic of biotinylated MDM2 peptides (B) used subsequently in (C) in vitro deacetylation assays of biotinylated MDM2 peptide incubated with lysates from 293T cells expressing the indicated Flag-SIRT. n = 2 biological replicates. (D) Western blotting analysis after immunoprecipitation with HA antibody in whole-cell lysate from 293T cells transfected with HA-MDM2, Myc-tagged p300, and/or the indicated Flag-SIRT and then treated with 5 mM NIC and 1 μM TSA. n = 2 biological replicates. (E) Western blotting analysis after pulldown of endogenous MDM2 from whole-cell lysate from U2OS cells infected with control [green fluorescent protein (GFP)–] or SIRT1-targeted short hairpin RNA (shRNA) and then treated with 5 mM NIC and 1 μM TSA. n = 2 biological replicates. (F) Western blotting analysis after immunoprecipitation with SIRT1 antibody in whole-cell lysate from MCF7 cells treated with 10 μM MG-132 and 5 mM NIC. n = 2 biological replicates. (G) Model of MDM2 acetylation regulated by p300 and SIRT1.

Fig. 3. MDM2 acetylation triggers p53 ubiquitination but blocks MDM2 self-ubiquitination

(A) Western blotting after pulldown by nickel–nitrilotriacetic acid (Ni-NTA) beads in whole-cell lysates from 293T cells transfected with wild-type (WT) or mutant His-Ub and HA-MDM2 and treated with 10 μM MG-132. n = 2 biological replicates. (B and C) Western blotting analysis of whole-cell lysate from Dox-inducible U2OS cells expressing WT or mutant MDM2 and treated with cycloheximide (CHX; 150 μg/ml) in the presence of Dox (1 μg/ml) (B). Quantification of signal intensity of HA-MDM2 normalized to that at t = 0 (C). Data are means ± SD from three independent experiments. *P < 0.05, unpaired Student’s t test. (D) Western blotting analysis after immunoprecipitation with MDM2 antibody in whole-cell lysate from U2OS cells infected with one of two SIRT1 shRNAs. n = 2 biological replicates. (E) Western blotting analysis after immunoprecipitation with Flag antibody in whole-cell lysate from 293T cells cotransfected with HA-MDM2 constructs and Flag-HAUSP and treated with 10 μM MG-132. n = 2 biological replicates. (F and G) Schematic of the MDM2 mutants (F) used subsequently in (G) Western blotting analysis of precipitates after pulldown by glutathione S-transferase (GST)–Sepharose beads in whole-cell lysate from 293T cells transfected with the GST-tagged MDM2 acidic region construct, HA-tagged HAUSP, and/or the GFP-tagged NLS domain. Lysates were pulled down with GST-Sepharose beads. The precipitates and whole-cell lysate was subjected to Western blotting analysis. n = 3 biological replicates. (H and I) Schematic of the MDM2 mutants (H) used subsequently in (I) Western blotting analysis of precipitates after pulldown by GST-Sepharose beads in whole-cell lysate from 293T cells transfected with HA-MDM2 and the indicated GST-tagged MDM2 acidic region construct. n = 2 biological replicates. (J) Schematic models of intramolecular regulation of MDM2 by acetylation to direct its E3 ubiquitin ligase activity toward itself or p53. (K) Western blotting analysis of precipitates after pulldown by Ni-NTA beads in whole-cell lysate from 293T cells cotransfected with WT or mutant HA-MDM2, His-Ub, and Flag-p53 and treated with MG-132. n = 2 biological replicates.

Fig. 4. Acetylation of MDM2 regulates its subcellular distribution

(A) Sequence alignment of MDM2, p27, FOXO1, and SKP2 to show sequence similarity in the nuclear localization sequence (NLS) region. (B) Acetylation of MDM2 promoted its cytoplasmic localization. Immunofluorescence analysis of HA staining (to assess MDM2 localization) in 293T cells transfected with HA-MDM2. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 20 μm. n = 2 biological replicates. (C) In vitro interaction of synthetic biotinylated MDM2 peptides acetylated or not at the Lys¹⁸² and/or Lys¹⁸⁵ residues with Importin α5 or Importin α7. n = 2 biological replicates. (D) Western blotting analysis after immunoprecipitation with Flag antibody in whole-cell lysate from 293T cells transfected with Flag–Importin α7 and/or HA-MDM2. n = 2 biological replicates.

Fig. 5. MDM2 acetylation destabilizes p53 and subsequently inhibits cellular apoptosis in response to DNA damage

(A) Western blotting analysis of whole-cell lysate from U2OS cells transfected with HA-MDM2 and either untreated or treated with 25 μM ETO for 24 hours. n = 2 biological replicates. (B) Western blotting analysis after immunoprecipitation with p53 antibody in whole-cell lysate from U2OS cells transfected with the indicated HA-MDM2 constructs and treated with 1 mM MG-132 and/or 25 μM ETO. n = 2 biological replicates. (C) Western blotting analysis after immunoprecipitation with HAUSP antibody in whole-cell lysate from U2OS cells infected with shRNA targeting GFP or SIRT1 and either untreated or treated with 25 μM ETO. n = 2 biological replicates. (D) Luciferase activity in Dox-inducible U2OS cells transfected with pGL3-puma-luc and treated with Dox (1 μg/ml) and/or 25 μM ETO for 24 hours. Data were normalized to luciferase activity from untreated cells and are presented relative to that in pGL3 vector–transfected controls. Data are means ± SD from three independent experiments. *P < 0.05, unpaired Student’s t test. DMSO, dimethyl sulfoxide. (E and F) Apoptosis assessed by flow cytometry in Dox-inducible U2OS cells treated with or without 25 μM ETO and stained for propidium iodide (E) or Annexin V (F). Data are means ± SD from three independent experiments. *P < 0.05, unpaired Student’s t test. (G) MTS assay for cell viability in Dox-inducible U2OS cells either untreated or treated with 25 μM ETO overnight in the presence of Dox (1 μg/ml). Data are means ± SD from three independent experiments. *P < 0.05, unpaired Student’s t test. (H) Colony formation in Dox-inducible U2OS cells treated with ETO in the presence of Dox and stained with crystal violet. Data are means ± SD from three independent experiments. *P < 0.05, unpaired Student’s t test. (I) Western blotting analysis after immunoprecipitation with MDM2 antibody in whole-cell lysate from U2OS cells treated with 15 μM MG-132 and 25 μM ETO. n = 2 biological replicates. (J) Western blotting analysis of whole-cell lysate from MCF7 cells infected with GFP- or SIRT1-shRNA and treated with NCS (100 ng/ml) for the indicated time (hours). n = 3 biological replicates. (K) Proposed model for acetylation-dependent regulation of MDM2 E3 ligase activity and substrate selectivity by p300. Under basal conditions, MDM2 is acetylated by p300 and then interacts with its upstream deubiquitinase HAUSP. HAUSP blocks MDM2 self-ubiquitination and stabilizes MDM2, subsequently resulting in p53 ubiquitination. On the other hand, under conditions of genotoxic stress, MDM2 is deacetylated by SIRT1, which triggers self-ubiquitination in part by its dissociation from HAUSP.