Abstract
Genetic studies have shown that under non-stressed conditions both MDM2 and MDMX are essential to maintain the tumor suppressor p53 in its latent form. It has also been shown that MDMX was rapidly degraded by MDM2 in response to DNA damage, a phenomenon which is critical for p53 activation. Thus, it is well documented that both MDM2 and MDMX play important roles for in the regulation of p53. However, the underlying molecular mechanisms of how these inhibitors cooperate and regulate p53 are still unclear. To gain insight into this question, we have studied using various chimeric proteins and demonstrated that MDM2 and MDMX can form a complex in vivo, and this heterocomplex had the ability to ubiquitinate p53 far more efficiently than MDM2 alone. Moreover, disruption of the binding between MDM2 and MDMX resulted in a marked increase in both abundance and activity of p53, emphasizing the functional importance of this heterocomplex in p53 control. In order to investigate further possible molecular mechanisms of the p53 regulation by MDM2 and MDMX, we are trying to develop an in vitro assay system in which ubiquitination studies of p53, MDM2 and MDMX can be performed. Here, we present data validating this assay system and report a novel mechanism by which the MDM2/MDMX heterocomplex regulates the p53 ubiquitination.
