A Single Conserved Amino Acid Residue as a Critical Context-Specific Determinant of the Differential Ability of Mdm2 and MdmX RING Domains to Dimerize

Mdm2 and MdmX are related proteins serving in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer as an E3 ubiquitin ligase for the tumor suppressor p53. The dimerization is required for the E3 activity and is mediated by the conserved RING domains present in both proteins, but only the RING domain of Mdm2 can form homodimers efficiently. We performed a systematic mutational analysis of human Mdm2, exchanging parts of the RING with the corresponding MdmX sequence, to identify the molecular determinants of this difference. Mdm2 can also promote MdmX degradation, and we identified several mutations blocking it. They were located mainly at the Mdm2/E2 interface and did not disrupt the MdmX-Mdm2 interaction. Surprisingly, some mutations of the Mdm2/E2 interface inhibited MdmX degradation, which is mediated by the Mdm2/MdmX heterodimer, but did not affect p53 degradation, mediated by the Mdm2 homodimer. Only one mutant, replacing a conserved cysteine 449 with asparagine (C449N), disrupted the ability of Mdm2 to dimerize with MdmX. When we introduced the cysteine residue into the corresponding site in MdmX, the RING domain became capable of forming dimers with other MdmX molecules in vivo, suggesting that one conserved amino acid residue in the RINGs of Mdm2 and MdmX could serve as the determinant of the differential ability of these domains to form dimers and their E3 activity. In immunoprecipitations, however, the homodimerization of MdmX could be observed only when the asparagine residue was replaced with cysteine in both RINGs. This result suggested that heterocomplexes consisting of one mutated MdmX RING with cysteine and one wild-type MdmX RING with asparagine might be less stable, despite being readily detectable in the cell-based assay. Moreover, Mdm2 C449N blocked Mdm2-MdmX heterodimerization but did not disrupt the ability of Mdm2 homodimer to promote p53 degradation, suggesting that the effect of the conserved cysteine and asparagine residues on dimerization was context-specific. Collectively, our results indicate that the effects of individual exchanges of conserved residues between Mdm2 and MdmX RING domains might be context-specific, supporting the hypothesis that Mdm2 RING homodimers and Mdm2-MdmX heterodimers may not be entirely structurally equivalent, despite their apparent similarity

Ectopic expression of TFII-I activates CMV promoter.

<p>(A) CMV promoter sequence with potential TFII-I binding sites. (B) Schematic representation of CMVdel luciferase vectors used in this study. (C) CMVdel series vectors were transfected into U2OS cells, either alone or in combination with pcDNA4/TFII-I and luciferase activity was measured in cell extracts 24 h post-transfection. The upper graph shows the relative changes in luciferase activity induced by TFII-I for each construct of the series individually. Data obtained in three independent experiments are presented as mean +/- standard deviation (* P<0.05, *** P<0.001). The obtained P values suggest significant differences in luciferase activity between cells co-transfected with TFII-I and cells transfected only with the respective luciferase construct. The lower graph shows the same set of results presented in relation to the full-length construct (activity in cells transfected with CMVdel1 was set as 1). Representative Western blot shows TFII-I protein levels in each sample (loaded in the same order as in the graphs). Endogenous PCNA served as a loading control.</p

Endogenous TFII-I contributes to CMV promoter activity in human cells.

<p>U2OS cells were transfected with a mixture of siRNAs targeting TFII-I (si TFII-I) or non-targeting control siRNAs (si ctrl). Twenty four hours later, the same cells were transfected again, this time with the CMVdel1 luciferase construct (A) or the β-galactosidase construct pcDNA3.1(-)/myc-His/LacZ (LacZ) (B). Cells were lysed 24 hours later. The graphs show the relative changes in luciferase activity or β-galactosidase activity, respectively, in three independent experiments (mean +/- standard deviation; * P<0.05, ** P<0.01). The representative results of Western blotting analysis illustrate the efficiency of TFII-I knock down in the presented experiments.</p

TFII-I enhances gene expression from plasmid constructs containing CMV promoter.

<p>Plasmid constructs coding for human oncogene Mdm2, human deubiquitinase USP48, human tumor suppressor p14 (Arf), jellyfish green fluorescent protein (GFP), and bacterial β-galactosidase (β-Gal) under the control of the CMV promoter were transfected into U2OS cells alone or in combination with pcDNA4/TFII-I. Whole cell extracts were resolved by SDS-PAGE and protein expression was analysed by Western blotting. Alpha-tubulin (α-Tub) served as a loading control.</p

Mdm2 over-expression does not lead to TFII-I degradation.

<p>(A) U2OS cells were transfected with plasmids coding for TFII-I (pcDNA4/TFII-I) and Mdm2 (pCHDM1A) and changes in TFII-I protein levels 24 hours post-transfection were analyzed using SDS-PAGE and Western blotting. PCNA served as a loading control. (B) U2OS cells were transfected with plasmids encoding TFII-I (pcDNA4/TFII-I) and Mdm2 (pCHDM1A), irradiated with ionizing radiation (γ rays, 6Gy) 24 hours post-transfection, and lysed 6 hours later. Western blotting was used to compare TFII-I protein levels in cell lysates of unirradiated controls and irradiated samples, both in the absence and in the presence of Mdm2 over-expression.</p

Effects of TFII-I and Mdm2 on transcription are specific to CMV promoter.

<p>(A) Over-expression of TFII-I and Mdm2 does not influence the activity of HIV 3’ LTR promoter. H1299 cells were transfected with pHIVlacZ (HIV-LacZ) alone or with HIV-LacZ plus pCEP4-Tat (TAT), together with plasmids coding for TFII-I (pcDNA4/TFII-I) and Mdm2 (pcHDM1A). The activity of β-galactosidase was measured 24 h later. Data obtained in three independent experiments are presented as mean +/- standard deviation. Representative Western blot shows TFII-I and Mdm2 protein levels in lysates of transfected cells. Endogenous PCNA served as a loading control. (B) Over-expression of TFII-I does not induce significant changes in cell proliferation or viability. U2OS cells were transfected with pEGFP-C2 to mark the transfected cells with GFP, together with either pcDNA4/LacZ (GFP+LacZ) or with pcDNA4/TFII-I (GFP+TFII-I). Flow cytometry was used 24 h post-transfection to determine the proportion of GFP-positive cells in the total cell population and cell viability in a PI exclusion assay. The experiment was performed in triplicates and the table shows the real counts of GFP-positive cells, their proportion in total cell population (mean) and the proportion of dead transfected cells (PI-positive, GFP-positive) in total cell population (mean). Untransfected U2OS cells served as a negative control. The Western blot shows GFP, TFII-I and Mdm2 expression in total cell lysates of transfected cells. (C) Sp1 does not require Mdm2 co-expression for inhibiting CMV promoter. H1299 cells were transfected with pcDNA4/LacZ alone, with pcDNA4/LacZ plus pcDNA4/TFII-I or pcDNA4/LacZ plus pEGFP-Sp1, together with plasmid coding for Mdm2 (pcHDM1A). The activity of β-galactosidase was measured 24 h later (right panel). Data obtained in three independent experiments are presented as mean +/- standard deviation</p

Mdm2 interacts with TFII-I.

<p>(A) HEK293T cells were transfected with a plasmid coding for human Mdm2. Endogenous TFII-I was co-immunoprecipitated from cell lysates using anti-Mdm2 antibody. (B) U2OS cells were transfected with a plasmid coding for human Mdm2. The subcellular localization of endogenous TFII-I and of the ectopically expressed Mdm2 was determined by immunofluorescence. DAPI was used to label cell nuclei. (C) Endogenous TFII-I and Mdm2 were co-immunoprecipitated from H1299 cell extracts using anti-TFII-I and anti-Mdm2 mouse monoclonal antibodies. Anti-tubulin α antibody served as a negative control. (D) Plasmids coding for GST and GST-tagged TFII-I mutant lacking the nuclear localization signal (ΔNLS) were transfected into U2OS cells either alone or together with Mdm2. Subcellular localization of the GST protein tag, GST-TFII-IΔNLS, and Mdm2 was analyzed using immunofluorescence. DAPI staining was used to label cell nuclei.</p