Functional analysis of a putative ATM/ATR substrate RFWD3

描述（申请人提供）：肿瘤抑制蛋白p53是细胞周期阻滞、细胞凋亡和基因组稳定性的关键调节因子。50%的人类癌症都发生了损害其功能的p53突变。在非应激细胞中，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连接酶泛素化并靶向降解。在基因毒性应激反应中，p53需要快速稳定以上调基因转录。至少有五种泛素连接酶（E6-AP、HDM2、ARF-BP1、COP1和PIRH2）和一种与这些连接酶相反的泛素特异性蛋白酶（HAUSP）被鉴定为介导泛素依赖的p53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损伤检查点是一种信号转导通路，在存在DNA损伤的情况下停止或延迟细胞周期进程。两个激酶，ATM和ATR，是这一途径的关键上游调节因子。在我们初步研究的新底物的蛋白质组学筛选中，我们发现无名指和WD重复结构域3 （RFWD3）蛋白是一种假定的ATM/ATR底物，是细胞周期阻滞所必需的。此外，我们发现RFWD3是p53在DNA损伤反应中稳定所必需的。我们假设RFWD3是一种新的E3连接酶，在哺乳动物DNA损伤反应网络中发挥重要作用，部分是作为p53稳定性的正调节因子。我们建议1)确定RFWD3调控DNA损伤检查点的机制；这是否通过调节p53泛素化和稳定性来实现；2)绘制RFWD3磷酸化位点，并测试ATM/ATR对其磷酸化如何调节其功能；3)在循环细胞和DNA损伤响应中分离rfwd3相关复合物，并通过质谱法鉴定其成分；我们的目标是找到并验证重要的RFWD3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。公共卫生相关性：这是一种新发现的假定的ATM/ATR底物，当被siRNA灭活时，会导致p53积累缺陷和细胞周期停滞。因此，RFWD3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p53新的正调节因子并了解其功能将有可能促进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.