The stabilization mechanism of mutant-type p53 by impaired ubiquitination:the loss of wild-type p53 function and the hsp90 association

Mutant-type p53 (mt p53) is largely accumulated in cancer cells due to its increased stability, To elucidate the mechanism of mt p53 stabilization, we analysed the turnover of p53 mutated at codon 248 whose alteration is most frequently found in human cancers. Proteasome inhibition induced the accum ulation of ubiquitinated mt p53, indicating that the ubiquitinated forms we re essentially unstable and degraded by the proteasome, The presence of a s mall amount of the ubiquitinated mt p53 relative to the abundant non-ubiqui tinated form suggested that the mt p53 ubiquitination was a rate-limiting p rocess in the slow turnover, Two phenomena destabilizing mt p53 via the ubi quitin-proteasome degradation were proved to be independent, First, the coe xpression of wild-type p53 (wt p53) promoted mt p53 destabilization as feed back regulation. Second, geldanamycin also induced mt p53 destabilization t hrough the dissociation of the protein from hsp90 but not through the resto ration of wt p53 function. Neither the mutant-specific conformation nor the N-terminal phosphorylation seemed to contribute directly to the mt p53 sta bilization, Further, a two-dimensional gel electrophoresis revealed that mo st of the post-translationally modified mt p53 was equally subjected to ubi quitination and subsequent proteasomal degradation, These findings are evid ence that mt p53 stabilization depends on the impaired ubiquitination due t o both the loss of wt p53 function and the hsp90 association.