P53 stabilization and activation in response to stress is critical for its tumor suppressor function. DNA damage induces p53 accumulation by activating ATM. We recently identified multiple novel ATM phosphorylation sites near the RING domain of MDM2, an E3 ligase that promotes p53 ubiquitination. Mutational analyses showed that these sites act in a redundant fashion to regulate p53 stability after DNA damage. Blocking MDM2 phosphorylation prevents p53 stabilization after DNA damage. Phosphorylation of MDM2 specifically blocks p53 poly-ubiquitination but not mono- ubiquitination. We also found that MDM2 interacts with histone methyltransferases and induces p53 C terminal lysine methylation. Therefore, MDM2 is an important signaling target in ATM stabilization of p53. Furthermore, MDM2 regulates p53 transcriptional activity through novel mechanisms. The following experiments are proposed to further study the mechanisms of p53 activation during stress response. (1) Determine the mechanism of p53 stabilization by MDM2 phosphorylation after DNA damage. (2) Investigate the mechanism of p53 stabilization by MDM2-binding proteins during non-genotoxic stress. (3) Investigate the regulation of p53 activity by MDM2-mediated lysine methylation. (4) Test the in vivo function of MDM2 phosphorylation in p53 tumor suppression. These experiments will lead to better understanding of the novel mechanisms that activate p53, and are critical for developing novel strategies of targeting MDM2 in cancer. PUBLIC HEALTH RELEVANCE: The proposal will study the regulation of the mdm2 oncoprotein and the mechanism of p53 inactivation by mdm2. The experiments are based on recent findings on the regulation of mdm2 activity by phosphorylation and histone methyltransferases. The experiments will also use mouse models to test the physiological functions of mdm2 phosphorylation. Understanding the mechanisms of mdm2 regulation may lead to new strategies to activate p53 in cancer cells.