RESUMEN
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.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Análisis Mutacional de ADN , Dimerización , Evolución Molecular , Células HEK293 , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/genéticaRESUMEN
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.