Functional analysis of a putative ATM/ATR substrate RFWD3

描述（由申请人提供）：肿瘤抑制蛋白p53是细胞周期停滞、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E3连接酶泛素化并被靶向降解。为了应对基因毒性应激，p53需要迅速稳定以上调基因转录。已经鉴定了至少五种泛素连接酶（E6-AP、HDM 2、ARF-BP 1、COP 1和PIRH 2）和对抗连接酶的泛素特异性蛋白酶（HAUSP）介导p53的泛素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稳定性，特别是在DNA损伤后尚不清楚。DNA损伤检查点是在存在DNA损伤的情况下停止或延迟细胞周期进程的信号转导途径。两种激酶ATM和ATR是该途径的关键上游调节剂。在我们的初步研究的新底物的蛋白质组学筛选中，我们发现，环指和WD重复结构域3（RFWD 3）蛋白是一个假定的ATM/ATR底物，需要细胞周期停滞。此外，我们发现RFWD 3是稳定p53在响应DNA损伤所必需的。我们推测RFWD 3是一种新型的E3连接酶，在哺乳动物DNA损伤反应网络中起着重要作用，部分通过作为p53稳定性的正调节剂。我们的主要工作是：1）确定RFWD 3调节DNA损伤检查点的机制，是否通过调节p53的泛素化和稳定性来实现; 2）定位RFWD 3的磷酸化位点，并检测ATM/ATR如何磷酸化RFWD 3来调节其功能; 3）分离RFWD 3相关复合物，并通过质谱鉴定其组成成分;我们的目标是发现和验证重要的RFWD 3调节因子及其E3连接酶底物，从而揭示其其他细胞功能的线索。公共卫生解放：这是一种新鉴定的推定ATM/ATR底物，当被siRNA灭活时，会导致p53积累和细胞周期停滞的缺陷。因此，RFWD 3可能在哺乳动物DNA损伤反应网络中发挥重要作用，作为一种新的E3连接酶，积极调节p53的稳定性。发现新的p53正调控因子并了解其功能可能会提高我们对癌症发展和治疗的认识。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVENCE: This 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.