Functional analysis of a putative ATM/ATR substrate RFWD3

项目描述 肿瘤抑制蛋白p53是细胞周期阻滞、凋亡和基因组稳定性的关键调节因子。 p53基因的突变损害了其功能，发生在50%的人类癌症中。在未应激的细胞中，p53是 维持在低水平;这是通过泛素-蛋白酶体系统实现的，其中p53被泛素化。 E3连接酶和靶向降解。为了应对遗传毒性应激，p53需要迅速稳定， 上调基因转录。至少五种泛素连接酶（E6-AP、HDM 2、ARF-BP 1、COP 1和PIRH 2）和 已经鉴定出对抗连接酶的泛素特异性蛋白酶（HAUSP）介导泛素- p53的依赖性蛋白酶体降解。然而，这些连接酶和蛋白酶如何协调调节 p53的稳定性，特别是DNA损伤后的稳定性尚不清楚。DNA损伤检查点是信号传导 在DNA损伤的情况下停止或延迟细胞周期进程的途径。两种激酶，ATM和 ATR是该途径的关键上游调节因子。在蛋白质组学筛选新底物， 初步研究发现，Ring finger and WD repeat domain 3（RFWD 3）蛋白是一种推定的 ATM/ATR底物是细胞周期阻滞所需的。此外，我们发现RFWD 3是必需的， p53对DNA损伤的反应。我们假设RFWD 3是一种新的E3连接酶， 在哺乳动物DNA损伤反应网络中的重要作用，部分是作为一个积极的调节剂， p53稳定性我们建议1）确定RFWD 3调节DNA损伤的机制 检查点;这是否是通过调节p53泛素化和稳定性实现的; 2）绘制RFWD 3 磷酸化位点，并测试ATM/ATR磷酸化如何调节其功能; 3）分离 RFWD 3相关复合物在循环细胞和响应DNA损伤，并确定其组成部分 我们的目标是发现和验证重要的RFWD 3调节剂及其E3连接酶底物， 从而揭示其其他细胞功能的线索。项目叙述 RFWD 3是一种新鉴定的推定ATM/ATR底物，当被siRNA灭活时， p53积累和细胞周期停滞。因此，RFWD 3可能在哺乳动物DNA中起重要作用 损伤反应网络作为一种新的E3连接酶，积极调节p53的稳定性。揭开 新的p53正调控因子，并了解其功能将有可能提高我们对癌症的认识 发展和治疗。

Project Description The tumor suppressor protein p53 is a key regulator of cell cycle arrest, apoptosis and genomic stability. Mutations of p53 that compromise its function occur in 50% of human cancers. In unstressed cells, p53 is maintained at low levels; this is achieved by the ubiquitin-proteasome system, in which p53 is ubiquitinated by E3 ligases and targeted for degradation. In response to genotoxic stress, p53 needs to be rapidly stabilized to upregulate gene transcription. At least five ubiquitin ligases (E6-AP, HDM2, ARF-BP1, COP1, and PIRH2) and an ubiquitin-specific protease (HAUSP) that opposes the ligases have been identified to mediate ubiquitin- dependent proteasomal degradation of p53. However, how these ligases and protease coordinate to regulate p53 stability, particularly after DNA damage is not clear. DNA damage checkpoints are signal transduction pathways that stop or delay the cell cycle progress in the presence of DNA damage. Two kinases, ATM and ATR, are key upstream regulators of this pathway. In a proteomic screen for novel substrates for our preliminary studies, we found that ring finger and WD repeat domain 3 (RFWD3) protein is a putative ATM/ATR substrate that is required for cell cycle arrest. Furthermore, we found that RFWD3 is required for stabilization of p53 in response to DNA damage. We hypothesize that RFWD3 is a novel E3 ligase that plays an important role in mammalian DNA damage response network in part by serving as a positive regulator of p53 stability. We propose to 1) determine the mechanism by which RFWD3 regulates DNA damage checkpoint; whether this is achieved by modulating p53 ubiquitination and stability; 2) to map the RFWD3 phosphorylation sites and test how its phosphorylation by ATM/ATR regulates its function; 3) to isolate RFWD3-associated complexes in cycling cells and in response to DNA damage and identify their components by mass spectrometry; we aim to find and validate important RFWD3 regulators and its E3 ligase substrates, thus revealing clues for its other cellular functions. Project Narrative RFWD3 is a newly identified putative ATM/ATR substrate that, when inactivated by siRNA, causes defects in p53 accumulation and cell cycle arrest. Thus, RFWD3 may play an important role in the mammalian DNA damage response network by serving as a novel E3 ligase that positively regulates p53 stability. Uncovering new positive regulator of p53 and understand its function will potentially advance our knowledge of cancer development and treatment.