The RING finger domain of MDM2 is essential for MDM2- mediated TGF-β resistance

In this study, we attempt to gain insights into the molecular mechanism underlying MDM2-mediated TGF-β resistance. MDM2 renders cells refractory to TGF-β by overcoming a TGF-β-induced G1 cell cycle arrest. Because the TGF-β resistant phenotype is reversible upon removal of MDM2, MDM2 likely confers TGF-β resistance by directly targeting the cellular machinery involved in the growth inhibition by TGF-β. Investigation of the structure-function relationship of MDM2 reveals three elements essential for MDM2 to confer TGF-β resistance in both mink lung epithelial cells and human mammary epithelial cells. One of these elements is the C-terminal half of the p53-binding domain, which at least partially retained p53-binding and –inhibitory activity. Secondly, the ability of MDM2 to mediate TGF-β resistance is disrupted by mutation of the nuclear localization signal, but is restored upon co-expression of MDMX, Finally, mutations of the zinc coordination residues of the RING finger domain abrogates TGF-β resistance, but not the ability of MDM2 to inhibit p53 activity or to bind MDMX. These data suggest that RING finger-mediated p53 inhibition and MDMX interaction are not sufficient to cause TGF-β resistance, and imply a crucial role of the E3 ubiquitin ligase activity of this domain in MDM2-mediated TGF-β resistance.