Exploring the in vivo role of the Mdm2 RING finger domain in p53 regulation.

摘要

Classically characterized as an "overarching" tumor suppressor, p53 is frequently found mutated, deleted, or misregulated in human cancers. Through its role as a transcription factor, p53 is capable of influencing the transcription of a number of genes critical in cell cycle arrest, senescence, apoptosis, and cellular metabolism. Mdm2 is widely accepted as the primary negative regulator of p53; canonically, Mdm2 is thought to regulate p53 through two mechanisms; 1) directly binding the p53 transactivation domain, suppressing p53 activity, and 2) functioning as an E3 ubiquitin ligase capable of ubiquitinating p53, targeting it for nuclear export and degradation by the proteasome. MdmX, a homologue and binding partner of Mdm2, is also capable of regulating p53 activity through directly binding to the p53 transactivation domain, however, MdmX does not demonstrate intrinsic E3 ligase activity. Both Mdm2:MdmX binding and Mdm2 E3 ligase function are conferred by the Mdm2 RING finger domain. To better understand the role of the Mdm2 RING finger domain in p53 regulation in vivo, we have generated a knock-in mouse model harboring a Tyr-to-Ala substitution at the 487 amino acid residue of murine Mdm2, Mdm2Y487A, which inactivates Mdm2 E3 ligase function without affecting its ability to bind with MdmX, effectively separating these two Mdm2 RING finger domain functions. Unexpectedly, Mdm2Y487A/Y487A mice were viable and developed normally into adulthood. While disruption of Mdm2 E3 ligase function by the Mdm2Y487A mutation resulted in p53 accumulation, p53 transcriptional activity remained low under basal conditions; however, exposure to even low levels of stress resulted in hyperactive p53, and p53-dependent mortality in Mdm2Y487A/Y487A mice. Disruption of Mdm2-mediated degradation of p53 serves as the underpinning of many cancer treatments aimed at reactivation of p53, but as observed in our Mdm2Y487A knock-in mouse, inactivation of Mdm2 E3 ubiquitin ligase function without obliterating Mdm2:MdmX interaction does not efficiently activate p53; however, disruption of Mdm2 E3 ligase function can increase sensitivity to irradiation induced DNA damage and lethality. This study provides further insight into the Mdm2-p53 autoregulatory loop and sheds new light on current issues surrounding the development of effective therapeutics targeting the p53 pathway.