Functional analysis of a putative ATM/ATR substrate RFWD3

假定的 ATM/ATR 基板 RFWD3 的功能分析

• 批准号: 7893391
• 负责人: YI WANG
• 金额: $ 11.51万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-31 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7893391
• 关键词: Affect; Antibodies; Apoptosis; Ataxia-Telangiectasia-Mutated protein kinase; BRCA1 gene; Binding; Biochemical; Cell Cycle; Cell Cycle Arrest; Cell physiology; Cells; Complex; DNA Damage; DNA damage checkpoint; Defect; Genetic Transcription; Genome Stability; Genotoxic Stress; Human; In Vitro; Knowledge; Ligase; MDM2 gene; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Molecular; Mutation; Pathway interactions; Peptide Hydrolases; Phosphorylation; Phosphorylation Site; Play; Polyubiquitin; Protein p53; Proteins; Proteomics; Ring Finger Domain; Role; Signal Pathway; Signal Transduction Pathway; Small Interfering RNA; System; TP53 gene; Testing; Tumor Suppressor Proteins; Ubiquitin; Ubiquitination; Up-Regulation; WD Repeat; cross reactivity; in vivo; multicatalytic endopeptidase complex; novel; oncoprotein p21; prevent; programs; response; therapy development; ubiquitin ligase; ubiquitin-protein ligase; ubiquitin-specific protease

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.

项目说明 抑癌蛋白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是 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被泛素化 E3连接酶和靶向降解。为了应对基因毒性应激，P53需要迅速稳定到 上调基因转录。至少五种泛素连接酶(E6-AP、Hdm2、ARF-BP1、COP1和PIRH2)和 一种与连接酶相反的泛素特异蛋白酶(Hausp)已被确定为介导泛素- 依赖蛋白酶体对P53的降解。然而，这些连接酶和蛋白酶是如何协同调节的 P53的稳定性，特别是DNA损伤后的稳定性尚不清楚。DNA损伤检查点是信号转导 在存在DNA损伤的情况下，停止或延缓细胞周期进展的途径。两个激活器，自动柜员机和 ATR是这一途径的关键上游调节因子。在蛋白质组筛选中寻找我们的新底物 初步研究发现，无名指和WD重复结构域3(RFWD3)蛋白是一种可能的 细胞周期停滞所需的ATM/ATR基板。此外，我们发现RFWD3是必需的 DNA损伤对P53的稳定作用。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，部分原因是作为一种正向调节因子 P53的稳定性。我们建议1)确定RFWD3调节DNA损伤的机制 检查点；这是否通过调节p53泛素化和稳定性来实现；2)定位RFWD3 并检测ATM/ATR对其功能的调节作用；3)分离 循环细胞中与RFWD3相关的复合体及其对DNA损伤的反应及其成分鉴定 我们的目标是寻找和验证重要的RFWD3调节子及其E3连接酶底物， 从而揭示了它的其他细胞功能的线索。项目叙事 RFWD3是一种新发现的ATM/ATR底物，当被siRNA灭活时，会导致 P53积聚和细胞周期停滞。因此，RFWD3可能在哺乳动物DNA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，从而破坏反应网络。揭开面纱 新的P53正向调节因子和对其功能的了解可能会促进我们对癌症的认识 发展和治疗。