MDM2 can promote the ubiquitination, nuclear export, and degradation of p53 in the absence of direct binding.

MDM2 can bind the N terminus of p53 and promote its ubiquitination and export from the nucleus to the cytoplasm, where p53 can then be degraded by cytoplasmic proteasomes. Several studies have reported that an intact MDM2 binding domain is necessary for p53 to be targeted for ubiquitination, nuclear export, and degradation by MDM2. In the current study, we examined whether the MDM2 binding domain of p53 could be provided in trans through oligomerization between two p53 molecules. p53 proteins mutated in their MDM2 binding domains were unable to bind MDM2 directly and were resistant to MDM2-mediated ubiquitination, nuclear export, and degradation when expressed with MDM2 alone. However, these same p53 mutants formed a complex with MDM2 and were efficiently ubiquitinated, exported from the nucleus, and degraded when co-expressed with MDM2 and wild-type p53. Moreover, this effect required MDM2 binding by wild-type p53 as well as oligomerization between wild-type p53 and the MDM2 binding-deficient p53 mutants. Taken together, these results support a model whereby MDM2 binding-deficient forms of p53 can bind MDM2 indirectly through oligomerization with wild-type p53 and are subsequently targeted for ubiquitination, nuclear export, and degradation. These findings may have important implications regarding the DNA damage response of p53.

Downregulation of MDM2 stabilizes p53 by inhibiting p53 ubiquitination in response to specific alkylating agents.

p53 is stabilized in response to DNA damaging stress. This stabilization is thought to result from phosphorylation in the N-terminus of p53, which inhibits p53:MDM2 binding, and prevents MDM2 from promoting p53 ubiquitination. In this report, the DNA alkylating agents mitomycin C (MMC) and methylmethane sulfonate (MMS), as well as UV radiation, stabilized p53 in a manner independent of phosphorylation in p53 N-terminus. This stabilization coincided with decreased levels of MDM2 mRNA and protein, and a corresponding decrease in p53 ubiquitination. Importantly, MDM2 overexpression inhibited the stabilization of p53 and decrease in ubiquitination following MMC, MMS, and UV treatment. This indicates that downregulation of MDM2 contributes to the stabilization of p53 in response to these agents.

MDM2-dependent ubiquitination of nuclear and cytoplasmic P53.

Wild-type p53 is stabilized and accumulates in the nucleus of DNA damaged cells. The effect of stabilizing p53 is to inhibit cell growth, either through a G1 cell cycle arrest or apoptotic cell death. MDM2 can inhibit p53 activity, in part, by promoting its rapid degradation through the ubiquitin proteolysis pathway. In the current study, MDM2-mediated degradation of p53 was partially inhibited in cells treated with leptomycin B (LMB), a specific inhibitor of nuclear export. In contrast, levels of ubiquitinated p53 increased in LMB-treated cells, indicating that nuclear export is not required for p53 ubiquitination. To investigate this further, p53 mutants were generated which localize to either the nucleus or cytoplasm, and their susceptibility to MDM2-mediated ubiquitination was assessed. p53 mutants that localized to either the nucleus or the cytoplasm were efficiently ubiquitinated, and their steady-state levels decreased, when coexpressed with MDM2. In addition, an MDM2-mutant that localized to the cytoplasm was able to ubiquitinate and degrade a p53 mutant which was similarly localized in the cytoplasm. Our results indicate that nuclear export is not required for p53 ubiquitination, and that p53 proteins that localize to either the nucleus or cytoplasm can be ubiquitinated and degraded by MDM2.

The MDM2 RING-finger domain is required to promote p53 nuclear export.

MDM2 can bind to p53 and promote its ubiquitination and subsequent degradation by the proteasome. Current models propose that nuclear export of p53 is required for MDM2-mediated degradation, although the function of MDM2 in p53 nuclear export has not been clarified. Here we show that MDM2 can promote the nuclear export of p53 in transiently transfected cells. This activity requires the nuclear-export signal (NES) of p53, but not the NES of MDM2. A mutation within the MDM2 RING-finger domain that inhibits p53 ubiquitination also inhibits the ability of MDM2 to promote p53 nuclear export. Finally, inhibition of nuclear export stabilizes wild-type p53 and leads to accumulation of ubiquitinated p53 in the nucleus. Our results indicate that MDM2-mediated ubiquitination, or other activities associated with the RING-finger domain, can stimulate the export of p53 to the cytoplasm through the activity of the p53 NES.

Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest.

p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.

Hypoxia induces p53 accumulation through MDM2 down-regulation and inhibition of E6-mediated degradation.

Hypoxia, a result of DNA-damaging agents such as ionizing radiation, induces the nuclear accumulation of the p53 tumor suppressor protein. However, unlike the effect in ionizing radiation, hypoxia readily induces the nuclear accumulation of p53 in HPV E6-infected cells. In HPV-infected cells, a key regulator of p53 protein levels is the E6 oncoprotein. In association with the endogenous cellular protein E6-associated protein (E6AP), E6 can accelerate the degradation of p53 under aerobic conditions. To better define the mechanism of p53 induction in E6-infected cells by hypoxia, we studied the expression and association of E6 and E6AP with p53 in vivo. We found that hypoxia did not alter the protein levels of E6 or E6AP as compared with those found under aerobic growth conditions, indicating that protein inhibition of E6 or E6AP alone is not sufficient to explain the increased accumulation of p53 under hypoxic conditions. However, p53 did fail to coprecipitate with E6AP under hypoxia, indicating that hypoxia uncouples the interaction of p53 with E6 and E6AP. We also present evidence to indicate that hypoxia decreases the expression of the endogenous cellular regulator of p53 protein, the human MDM2 protein, resulting in an inhibition of p53 export from the nucleus to the cytoplasm for degradation. Taken together, these results suggest that the hypoxic induction of p53 is attributable to the down-regulation of MDM2 protein levels and uncoupling of p53 from its interaction with the E6/E6AP complex.