RESUMO
The exact role of the central acidic domain of Mdm2 in p53 degradation remains unclear. We therefore performed a systematic and comprehensive analysis of the acidic domain using a series of short deletions and found that only a minor part of the domain was indispensable for Mdm2-mediated p53 ubiquitylation. Moreover, we identified a short stretch of acidic amino acids required for p53 degradation but not ubiquitylation, indicating that, in addition to p53 ubiquitylation, the acidic domain might be involved in a critical post-ubiquitylation step in p53 degradation. Rather than representing a single functional domain, different parts of the acidic region perform separate functions in p53 degradation, suggesting that it might be possible to therapeutically target them independently.
Assuntos
Análise Mutacional de DNA , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Proteína Supressora de Tumor p53/química , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Deleção de Genes , Células HEK293 , Humanos , Imunoprecipitação , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/química , Homologia de Sequência de Aminoácidos , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/químicaRESUMO
Mdm2 can mediate p53 ubiquitylation and degradation either in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer. The ubiquitin ligase activity of these complexes resides mainly in their respective RING finger domains and also requires adjacent C-terminal tails. So far, structural studies have failed to show significant differences between Mdm2 RING homodimers and Mdm2/MdmX RING heterodimers. Here, we report that not only the primary amino acid sequence, but also the length of the C-terminal tail of Mdm2 is highly conserved through evolution and plays an important role in Mdm2 activity toward p53. Mdm2 mutants with extended C termini do not ubiquitylate p53 despite being capable of forming Mdm2 homodimers through both RING-acidic domain and RING-RING interactions. All extended mutants also retained the ability to interact with MdmX, and this interaction led to reactivation of their E3 ubiquitin ligase activity. In contrast, only a subset of extended Mdm2 mutants was activated by the interaction with Mdm2 RING domain, suggesting that Mdm2 homodimers and Mdm2/MdmX heterodimers may not be structurally and functionally fully equivalent.
