Functional analysis of a putative ATM/ATR substrate RFWD3

描述(申请人提供)：肿瘤抑制蛋白P53是细胞周期停滞、细胞凋亡和基因组稳定的关键调节因子。破坏其功能的p53突变发生在50%的人类癌症中。在非应激细胞中，P53被维持在低水平；这是通过泛素-蛋白酶体系统实现的，在该系统中，P53被E3连接酶泛素化并被降解。为了应对遗传毒性应激，需要迅速稳定P53以上调基因转录。至少有5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)已被鉴定为介导泛素依赖的蛋白酶体对p53的降解。然而，这些连接酶和蛋白酶如何协调来调节P53的稳定性，特别是在DNA损伤后，目前还不清楚。DNA损伤检查点是在DNA损伤存在时停止或延缓细胞周期进程的信号转导通路。ATM和ATR这两个激酶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筛选中，我们发现RING FING和WD重复结构域3(RFWD3)蛋白是一种假定的ATM/ATR底物，是细胞周期停滞所必需的。此外，我们发现RFWD3是稳定P53对DNA损伤的反应所必需的。我们推测，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，部分原因是作为P53稳定性的正向调节因子。我们建议1)确定RFWD3调节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磷酸化位点的图谱，并测试其被ATM/ATR磷酸化如何调节其功能；3)分离周期细胞中与RFWD3相关的复合体，并通过质谱学鉴定其组分；我们的目标是寻找和验证重要的RFWD3调节器及其E3连接酶底物，从而揭示其其他细胞功能的线索。公共卫生研究进展：这是一种新发现的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可能通过作为一种新的E3连接酶在哺乳动物DNA损伤反应网络中发挥重要作用，正向调节P53的稳定性。发现新的P53正向调节因子并了解其功能，可能会促进我们对癌症发展和治疗的了解。

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.