Mechanism of NF-kappaB Activation by DNA Damage

描述(申请人提供)：核转录因子核因子-KB被DNA破坏的抗癌剂激活，已成为恶性行为的重要调节器，包括抵抗细胞凋亡。这一信号转导途径也是理解核DNA损伤如何诱导信号通路激活细胞质隔离的转录因子--核质信号转导通路的一个有吸引力的范例。这项建议的长期目标是增加我们对模式DNA损伤抗癌药物喜树碱和依托泊苷启动的核因子-KB信号转导通路的了解。在我们已发表的研究中，我们提供了证据表明，这些遗传毒性因素诱导了两条平行通路的汇聚：(A)依赖于DSB的ATM激活；以及(B)压力信号通过SUMO-1修饰导致kB激酶(Ikk)复合体的调节亚单位Nemo/Ikky的核积聚。最终，NEMO以ATM和泛素依赖的方式输出，并激活细胞质IKK复合体。因此，NEMO代表了一种共同的核信号，它介导了由这些不同的抗癌药物诱导的核质信号通路。通过以下特定的目的，我们将检验这一假设，即一系列NEMO翻译后修饰介导其核输入和输出到伴侣从细胞核到细胞质的IKK调节因子，以促进IKK的激活，以响应遗传毒性刺激。目的1：阐明基因毒性药物对NEMO基因SUMO-1的修饰机制。目的2：确定泛素在NEMO核输出基因毒性信号中的作用。目的3：阐明ATM在基因毒物激活IKK中的作用。拟议的研究将有助于确定某些具有遗传毒性的抗癌药物激活核因子-KB的基本机制。这些机制也可以作为其他遗传毒性物质，如电离辐射，以及其他靶向DNA拓扑异构酶I或II的试剂诱导的激活途径的模型。此外，新的试剂工具，如磷酸特异性NEMO抗体，编码野生型和突变型NEMO和泛素连接酶的cDNA，以及含有这些突变蛋白的细胞系统，也将对其他研究人员努力剖析NF-KB激活途径有用。最后，参与这一激活途径的特定酶和过程可能作为合理的治疗靶点，在某些类型的人类癌症中诱导化疗/放射增敏。

DESCRIPTION (provided by applicant): Activation of the transcription factor NF-KB by DNA-damaging anticancer agents has emerged as an important modulator of malignant behaviors, including resistance to apoptotic cell death. This signal transduction pathway also serves as an attractive paradigm to understand how nuclear DNA damage induces a signaling pathway to activate a cytoplasmically sequestered transcription factor - a nuclear-to-cytoplasmic signal transduction pathway. The long-term goal of this proposal is to increase our understanding of the mechanisms involved in NF-KB signal transduction pathways initiated by the model DNA-damaging anticancer agents, camptothecin and etoposide. In our published studies, we provided evidence that a convergence of two parallel pathways is induced by these genotoxic agents: (a) DSB-dependent activation of ATM; and (b) a stress signal that leads to nuclear accumulation of NEMO/IKKy, the regulatory subunit of the kB kinase (IKK) complex, via a SUMO-1 modification. Ultimately, NEMO exports in an ATM- and ubiquitin-dependent manner and activates the cytoplasmic IKK complex. Thus, NEMO represents a common nuclear signal that mediates the nuclear-to-cytoplasmic signaling pathway induced by these distinct anticancer agents. Through the following Specific Aims, we will test the hypothesis that a series of NEMO posttranslational modifications mediates its nuclear import and export to chaperone an IKK regulator from the nucleus to the cytoplasm to promote IKK activation in response to genotoxic stimuli. Aim 1: Specify the mechanism of SUMO-1 modification of NEMO by genotoxic agents. Aim 2: Determine the role of ubiquitin in nuclear export of NEMO in genotoxic signaling. Aim 3: Elucidate the role of ATM in IKK activation by genotoxic agents. The proposed studies will help determine the mechanisms fundamental to NF-KB activation by certain genotoxic anticancer agents. These mechanisms may also serve as models for activation pathways induced by other genotoxic agents, such as ionizing radiation, and other agents that target DNA topoisomerase I or II. Moreover, new reagent tools, such as phospho-specific NEMO antibodies, cDNAs encoding wild type and mutant versions of SUMO and ubiquitin ligases for NEMO, and cell systems that harbor these mutant proteins, will also be useful for other investigators in their efforts to dissect NF-KB activation pathways. Finally, specific enzymes and processes involved in this activation pathway may serve as rational therapeutic targets to induce chemo/radio-sensitization in certain types of human cancer.