Figure 1.
MDM2-ALT1 and MDMX-ALT2 interact with full-length MDM2 and MDMX.
A. Full-length MDM2 is encoded by exons 3 to 12 of the MDM2 gene and consists of the N-terminal p53-binding domain, the nuclear localization (NLS) and export signals (NES), the central ARF binding and Zinc finger domains and the C-terminal RING domain. MDM2-ALT1 comprises only exons 3 and 12 spliced together and the protein lacks the p53-binding domain. However, it retains the RING domain. B. Full-length MDMX, a close family member of MDM2, also comprises an N-terminal p53-binding domain, a central Zinc finger domain and a C-terminal RING domain and is encoded by exons 2 to 11 of the MDMX gene. MDMX-ALT2 consists of exons 2,3,10 and 11 and the protein is architecturally similar to MDM2-ALT1 in that it lacks the p53-binding domain but retains the RING domain. C. Myc-tagged constructs of LacZ, MDM2-ALT1 or MDMX-ALT2 were transfected into MCF7 cells. Immunoprecipitation of the myc-tagged proteins revealed the specific binding of full-length MDM2 to MDM2-ALT1 and MDMX-ALT2 and not to negative control protein myc-LacZ (compare lanes 2 and 3 to lane 1). Experiments were repeated a minimum of three times and consistent results were observed. Representative gel images are presented in the figure. D. Myc-tagged MDM2-ALT1 and MDMX-ALT2 co-immunoprecipitate with full-length MDMX while the negative control protein myc-LacZ does not interact with MDMX (compare lanes 2 and 3 to lane 1). These results were observed in two independent trials and representative images are shown.
Figure 2.
MDM2-ALT1 and MDMX-ALT2 expression causes upregulation of p53 and its downstream target p21.
A. The over-expression (O/E cell lysates) of the myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was confirmed using anti-myc tag antibody in the MCF7 cells that were transfected with the corresponding expression constructs (top panel). The level of p53 protein was examined in these samples using the anti-p53 antibody and an upregulation of p53 protein was observed upon MDM2-ALT1 or MDMX-ALT2 over-expression compared to LacZ expressing cells although the increase is more modest in MDMX-ALT2 over-expression (lanes 2 and 3 compared to lane 1). The positive control, UVC (50 J/m2) irradiated MCF7 cells show a strong upregulation of p53 protein levels in response to the stress when compared to untreated cells (lanes 4 and 5). β-actin was used as loading control. A minimum of three independent experiments was performed and representative gel images are shown. B. p21 expression at the mRNA level was examined using quantitative real-time PCR and GAPDH levels were used as the endogenous control. The ratio of p21 to GAPDH is represented graphically and the error bars represent standard deviations from at least 3 independent experiments. MCF7 cells over-expressing MDM2-ALT1 (2Alt1) show statistically significant increase in p21 transcript levels compared to LacZ expressing cells (p<0.01). The cells expressing MDMX-ALT2 (XAlt2) did not show statistically significant changes in p21 expression at the mRNA level. C. The levels of p21 protein in the MCF7 cells transfected with myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was examined using anti-p21 antibody. Both MDM2-ALT1 and MDMX-ALT2 over-expression lead to upregulation of p21 protein levels compared to LacZ over-expression (compare lanes 2 and 3 with lane 1). A minimum of three independent experiments was performed and consistent results observed. Representative images are shown here. Additionally, UVC-irradiated MCF7 cells were used as positive control and show an upregulation of p21 compared to untreated cells (lanes 4 and 5).
Figure 3.
MDM2-ALT1 over-expression leads to G1 phase cell cycle arrest in a p53-dependent and p21-dependent manner.
A. HCT116 wild-type (wt), B. HCT116 p53−/− and C. HCT116 p21−/− cells were transfected with myc-tagged GFP or MDM2-ALT1 (2Alt1) or MDMX-ALT2 (XAlt2), harvested 24 hours post-transfection and stained with propidium iodide solution and sorted for DNA content. The bar graphs represent the percentage of cells in the various phases of the cell cycle. Error bars represent the standard error mean from at least 3 independent experiments. HCT116 cells that are wildtype (A) show significantly higher percentage of cells in G1-phase upon MDM2-ALT1 expression (31.44% ±2.45 SEM) compared to GFP-expressing cells (24.40% ±1.40 SEM; n = 5, p = 0.0369). In case of MDMX-ALT2 over-expression in HCT116 wildtype cells, there is no significant change in percentage of cells in G1 phase compared to negative control GFP-expressing cells (p = 0.4389). HCT116 cells that are null for p53 (B) or p21 (C) do not show any differences in the percentage of cells in any of the cell cycle phases upon over-expression of GFP or MDM2-ALT1 or MDMX-ALT2. D. Representative immuno blot showing expression of p53, p21 and loading control GAPDH in HCT116 wt, p53−/− and p21−/− cells.
Figure 4.
MDM2-ALT1 and MDMX-ALT2 lead to the activation of subtly different subsets of p53 transcriptional targets.
A. MCF7 cells transfected with myc-tagged LacZ, or MDM2-ALT1 (Alt1) or MDMX-ALT2 (XAlt2) were examined for mRNA levels of a panel of 9 p53 transcriptional targets using quantitative real-time PCR and were normalized to LacZ-expressing cells. The bar graphs represent the relative quantification (2−ΔΔCt) values from at least 3 independent experiments and each consisting of 3 technical replicates. The error bars represent standard error means (SEM). B. qRT-PCR was used to compare the relative levels (2−ΔΔCt) of miRNA targets of p53 between cells over-expressing LacZ or MDM2-ALT1 or MDMX-ALT2. Here also a minimum of 3 independent trials was performed with 3 technical replicates each. The error bars represent standard error means (SEM). * represents p<0.05 and ** represents p<0.01 in all cases.
Figure 5.
Proposed model: MDM2-ALT1 and MDMX-ALT2 antagonize their full-length counterparts and lead to p53 stabilization.
A. Under normal conditions, MDM2 and MDMX function to maintain low levels of p53 (via ubiquitination and subsequent degradation) and curb its transcriptional activity by binding p53. This helps maintain homeostasis and normal cellular functions including cell cycle progression. B. Under genotoxic stress, alternative splice forms MDM2-ALT1 and MDMX-ALT2 interact with the full-length MDM proteins and interfere in their p53-regulatory functions. This leads to the stabilization and upregulation of p53 levels and also the activation of p53 transcriptional targets leading to changes in cell cycle progression.