Abstract
The tumor suppressor gene product p53 is one of the major determinants of cellular responses to ionizing radiation. It is well known from mouse genetic studies that both an E3 ubiquitin ligase MDM2 and its structural homologue MDMX play crucial roles in the p53 regulation. However, the underlying molecular mechanisms of how MDM2 and MDMX cooperate and regulate p53 function are still unclear. In order to investigate the molecular mechanism by which P53 is regulated by MDM2 and MDMX, we have established an in vitro p53-ubiquitination assay system. Using the assay system, we found out more about how P53 is ubiquitinated. Our findings provide novel insights into the potential role of p53 ubiquitination mediated by MDM2 and MDMX. Our results indicate that a requirement for MDMX in absolute ubiquitination of P53 by MDM2, and the complex formation of MDM2 and MDMX is necessary for the enhancement of MDM2's E3 ubiquitin ligase activity. Furthermore, we have shown that the amount of MDMX protein significantly affects on the oscillation pattern of P53 and MDM2, which determines the cellular fate in response to radiation exposure. Further studies of the role of MDM2 and MDMX would reveal the precise molecular mechanism of p53-mediated cellular responses to ionizing radiation.
