Abstract 3683: The stress response mediator ATF3 stabilizes p53 by competing with it for MDM2-mediated ubiquitination

Abstract

Abstract While the tumor suppressor p53 is crucial for the maintenance of genetic integrity upon DNA damage and oncogenic stresses, its level and activity are tightly controlled by a complex network largely mediated by the E3 ubiquitin ligase MDM2. MDM2 catalyzes the addition of ubiqutin chains to the C-terminus of p53 and is the major ubiquitin ligase to promote proteosomal degradation of p53. We previously demonstrated that ATF3, a common stress response mediator, can bind p53 and prevent it from MDM2-mediated ubiquitination. As a consequence, ATF3 can stabilize p53 upon DNA damage and prevent cells from oncogene-induced transformation by promoting p53-mediated senescence, in line with accumulating evidence supporting that ATF3 can act as a tumor suppressor. Interestingly, ATF3 is also a bona fide substrate of MDM2, and thus, the ATF3-MDM2 interaction can fine tune p53 activity in the DNA damage response. However, the mechanism by which ATF3 blocks p53 ubiquitination to activate the latter protein remains largely undefined. Efficient ubiquitination of p53 requires the binding of MDM2 to the N-terminus of p53, and the MDM2 binding to MDMX. Here we show that neither the p53-MDM2 interaction nor the MDM2-MDMX interaction is disrupted by the binding of ATF3 to p53 or MDM2. As both ATF3 and p53 are substrates of MDM2, we explored a possibility that ATF3 competes with p53 for MDM2-mediated ubiquitination. By in-vitro ubiquitination assays, we identified that the 5 lysine residues clustering in aa 100-115 of ATF3 are required for MDM2-mediated ubiquitination. Site-specific mutagenesis indicates that MDM2 mainly ubiquitinates ATF3 at lysine 107 (K107) and lysine 108 (K108) as MDM2 fails to add ubiquitin chains to ATF3 when these two residues were mutated to arginine. Importantly, while mutations at these two residues do not affect the ATF3 binding to MDM2 or p53, the ATF3 mutant devoid of ubiquitination fails to stabilize p53 and prevent p53 from MDM2-mediated ubiquitination. These results thus demonstrate that ATF3 can activate p53 by competing with the tumor suppressor for MDM2-mediated ubiquitination. Citation Format: Xingyao Li, Chunhong Yan. The stress response mediator ATF3 stabilizes p53 by competing with it for MDM2-mediated ubiquitination. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3683.