Mechanisms of p53 activation during stress response

描述（申请人提供）：P53在应激反应中的稳定和激活对其抑瘤功能至关重要。DNA损伤通过激活ATM诱导p53积累。我们最近在MDM2（一种促进p53泛素化的E3连接酶）的RING结构域附近发现了多个新的ATM磷酸化位点。突变分析表明，这些位点在DNA损伤后以冗余的方式调节p53的稳定性。阻断MDM2磷酸化可阻止DNA损伤后p53的稳定。MDM2的磷酸化特异性地阻断p53的多泛素化而非单泛素化。我们还发现MDM2与组蛋白甲基转移酶相互作用并诱导p53 C末端赖氨酸甲基化。因此，MDM2是p53 ATM稳定化的重要信号靶点。此外，MDM2通过新的机制调节p53的转录活性。为了进一步研究应激反应中p53的激活机制，我们拟开展以下实验。(1)确定DNA损伤后MDM2磷酸化稳定p53的机制。(2)探讨非基因毒性应激下mdm2结合蛋白稳定p53的机制。(3)探讨mdm2介导的赖氨酸甲基化对p53活性的调节作用。(4)检测MDM2磷酸化对p53肿瘤抑制的体内功能。这些实验将有助于更好地了解激活p53的新机制，并对开发针对MDM2的癌症新策略至关重要。

DESCRIPTION (provided by applicant): 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.