MDM2-dependent ubiquitination of nuclear and cytoplasmic P53

Wild-type p53 is stabilized and accumulates in the nucleus of DNA damaged c ells. The effect of stabilizing p53 is to inhibit cell growth, either throu gh a G1 cell cycle arrest or apoptotic cell death. MDM2 can inhibit p53 act ivity, in part, by promoting its rapid degradation through the ubiquitin pr oteolysis pathway, In the current study, MDM2-mediated degradation of p53 w as partially inhibited in cells treated with leptomycin B (LMB), a specific inhibitor of nuclear export. In contrast, levels of ubiquitinated p53 incr eased in LMB-treated cells, indicating that nuclear export is not required for p53 ubiquitination, To investigate this further, p53 mutants were gener ated which localize to either the nucleus or cytoplasm, and their susceptib ility to MDM2-mediated ubiquitination was assessed. p53 mutants that locali zed to either the nucleus or the cytoplasm were efficiently ubiquitinated, and their steady-state levels decreased, when coexpressed with MDM2, In add ition, an MDM2-mutant that localized to the cytoplasm was able to ubiquitin ate and degrade a p53 mutant which was similarly localized in the cytoplasm , Our results indicate that nuclear export is not required for p53 ubiquiti nation, and that p53 proteins that localize to either the nucleus or cytopl asm can be ubiquitinated and degraded by MDM2.