Identification of 14-3-3 binding site on MDMX. (A) Location of phosphorylation sites on MDMX. Phosphorylation of S342, S367, and S403 was detected by mass spectrometry and verified by phosphorylation-specific antibody analyses in previous studies. (B) MDMX mutant binding to 14-3-3. H1299 cells were transiently cotransfected with MDMX mutant and FLAG-14-3-3τ plasmids for 48 h, followed by anti-FLAG IP and MDMX Western blot. (C) 14-3-3 binding by phosphorylated S367 peptide. MDMX peptides with or without phosphorylation on S342 and S367 were chemically crosslinked to agarose beads. The beads were incubated with whole-cell extract, washed, and bound 14-3-3τ detected by Western blot.

Chk2 is required for DNA damage-induced phosphorylation of S367 and binding to 14-3-3. (A) Wild-type and Chk2-null HCT116 cells were treated with 10 Gy γ irradiation or 20 J/m² for 4 h in the presence of 30 μM MG132 to preserve phosphorylated MDMX. MDMX was immunoprecipitated with 8C6 and analyzed using anti-PS367 antibody by Western blot. The membrane was reprobed with 8C6 to confirm the level of total MDMX. (B) HCT116 cells were stably infected with MDMX lentivirus to facilitate detection of MDMX binding to endogenous 14-3-3. Cells were treated with 10 Gy γ irradiation for 4 h in the presence of 30 μM MG132. MDMX was immunoprecipitated and analyzed using anti-PS367 antibody (top panel). H1299 cells with very low MDMX level were used as negative control. MDMX immunoprecipitate was analyzed for the co-precipitation of 14-3-3τ.

S367 phosphorylated MDMX preferentially localizes to the nucleus. (A) HeLa cells stably transfected with MDMX were treated with 0.5 μM CPT for 18 h and stained using 8C6 or PS367 antibody. (B) 14-3-3 expression promotes elimination of phosphorylated MDMX from the nucleus. U2OS cells were transiently cotransfected with MDMX and 14-3-3τ plasmids for 24 h and treated with 30 μM MG132 for 4 h. Cells were double stained using 8C6 for total MDMX (green) and PS367 antibody for phosphorylated MDMX (red).

Chk2 promotes ubiquitination and degradation of MDMX through modification of the 14-3-3 binding site. (A) HCT116-Chk2−/− cells were transiently transfected with MDMX, MDM2, Chk2, and His6-ubiquitin. MDMX ubiquitination was detected by Ni-NTA purification, followed by MDMX Western blot. Whole-cell extract was analyzed for MDMX level by Western blot. Chk2-A347 is a kinase-deficient mutant. (B) HCT116-Chk2−/− cells were transfected with His6-ubiquitin, MDMX, 14-3-3τ, and Chk2 plasmids. MDMX ubiquitination was detected by Ni-NTA purification, followed by MDMX Western blot. 14-3-3ΔN is a 30–245 fragment of 14-3-3τ lacking the dimerization domain. (C) 14-3-3 stimulates degradation of PS367 MDMX. H1299 cells were transiently transfected with MDMX, MDM2, and 14-3-3τ for 48 h. MDMX was immunoprecipitated with 8C6 and probed with PS367 antibody for the phosphorylated form, or with 8C6 for total MDMX level.

MDMX forms a complex with 14-3-3. (A) Copurification of 14-3-3 with MDMX. HeLa cells stably transfected with FLAG-MDMX were immunoprecipitated using anti-FLAG M2 antibody, the purified MDMX and associated proteins were detected by Coomassie stain. The two marked bands were identified as CK1α and 14-3-3τ by mass spectrometric peptide sequencing. (B) Interaction of endogenous 14-3-3 and MDMX. MCF7 cells were treated with 30 μM MG132 for 4 h, 0.5 μM camptothecin for 16 h, or irradiated with 10 Gy γ irradiation for 4 h. Cell lysates were immunoprecipitated with MDMX antibody 8C6, followed by anti-14-3-3τ Western blot. (C) Interaction of MDMX with 14-3-3 isoforms. H1299 cells transiently cotransfected with MDMX and epitope-tagged 14-3-3 plasmids were immunoprecipitated using anti-FLAG (for τ, σ, γ), anti-myc (for ζ, β), and anti-His6 (for ɛ, η) antibodies.

S367 is a major MDMX phosphorylation site in vivo. 293T cells were transiently transfected with MDMX mutants with serine-to-alanine mutations at the indicated phosphorylation site and labeled with ³²P-orthophosphate. The labeled MDMX was immunoprecipitated, digested with Asp-N endoproteinase, and analyzed by 2D peptide mapping. (A) Wild-type MDMX. (B, C) MDMX point mutants.

Chk2 phosphorylation of MDMX is required for nuclear import and degradation. (A) S367A mutant does not undergo nuclear translocation after DNA damage. U2OS cells stably transfected with wild-type MDMX or S367A mutant were treated with 0.5 μM CPT for 18 h and stained using 8C6. (B) DNA damage does not induce MDMX nuclear import in Chk2-null cells. HCT116 wild-type and Chk2-null cells stably infected with lentivirus expressing MDMX were treated with 10 Gy irradiation for 18 h and stained with 8C6 antibody. (C) Analysis of endogenous MDMX distribution by subcellular fractionation. HCT116 cells were treated with 10 Gy γ irradiation in the presence or absence of 30 μM MG132 for 4 h. Nuclear and cytoplasmic fractions were analyzed by Western blot. To facilitate comparison of ratio, loading of IR-treated C/N pair was empirically increased to obtain MDMX signal similar to the untreated pair. (D) Chk2-null cells were partially defective for MDMX degradation. HCT116 cells were treated with indicated doses of γ irradiation for 4 h and analyzed by Western blot for different markers.

MDMX nuclear import after DNA damage requires a cryptic NLS. (A) GFP–MDMX fusions were stably transfected into U2OS cells and photographed for localization using fluorescence from the GFP tag. (B) Full-length MDMX with the K468E mutation was transiently transfected into U2OS cells, treated for 18 h with 0.5 μM CPT, and stained using 8C6 antibody. (C) GST-importin-loaded beads were incubated with lysate of HCT116 cells transiently transfected with wild-type or mutant MDMX. Bound MDMX were detected by Western blot. (D) Diagram of MDMX indicating the location of the cryptic NLS.

Chk2 and 14-3-3 cooperate to neutralize MDMX inhibition of p53. (A) HCT116-Chk2−/− cells were transfected with p53-responsive BP100-luc reporter to detect endogenous p53 transcriptional activity. The effects of wild-type MDMX, 14-3-3 and Chk2 coexpression on p53 activity were detected by luciferase assay and normalized to CMV-lacZ transfection efficiency control. (B) A control experiment identical to (A), except that the MDMX 367A mutant was used.