Fig 6. Mutant p53 inhibits ASK1-dependent phosphorylation of Daxx and prevents Daxx accumulation induced by TNFα.

(a) Mutant p53 inhibits ASK1-dependent phosphorylation of Daxx. HeLa cells were transfected with expression plasmids, including wild type and mutant p53 plasmids, in the indicated combinations. Thirty hours after transfection, cell extracts were subjected to immunoprecipitation with anti-phospho-Daxx specific antibody followed by western blotting with anti-Daxx antibody. Since the expression level of Daxx was reduced by the presence of mutant p53 (18), the amounts of cell lysate in the immunoprecipitation reactions were adjusted to contain equal amount of Daxx protein (WB: Daxx). (b) TNFα does not induce the accumulation of Daxx in cells expressing natural tumorigenic mutant p53. Two cell lines with wild type p53 (ME180 and HeLa) and three cell lines with expressing mutant p53 (HT29, A431, and A2058) were examined for the accumulation of Daxx in response to TNFα treatment. Cells were incubated with TNFα (0, 1, 5 ng/ml) for 18 h and the cell extracts were subjected to western blotting for Daxx and actin. (c) Depletion of mutant p53 restores TNFα-induced accumulation of Daxx and apoptosis. HT29 cells were infected (m.o.i. = 200) with adenoviruses encoding control or p53 shRNA for 5 days followed by the treatment with TNFα at the indicated concentration for 18 h. The cell extracts were analyzed by western blotting with indicated antibodies.