Mechanism of NF-kappaB Activation by DNA Damage

描述（由申请人提供）：dna损伤抗癌药物激活转录因子NF-KB已成为恶性行为的重要调节剂，包括对凋亡细胞死亡的抵抗。这种信号转导途径也可以作为一个有吸引力的范例来理解核DNA损伤如何诱导信号通路激活细胞质隔离的转录因子-核到细胞质的信号转导途径。这项提议的长期目标是增加我们对由模型dna损伤抗癌药物喜树碱和石甲苷启动的NF-KB信号转导途径的机制的理解。在我们发表的研究中，我们提供的证据表明，两个平行通路的收敛是由这些基因毒性药物诱导的：(a)依赖dsb的ATM激活；(b)胁迫信号，通过SUMO-1修饰导致kB激酶（IKK）复合物的调控亚基NEMO/IKKy的核积累。最终，NEMO以ATM和泛素依赖的方式输出并激活细胞质IKK复合物。因此，NEMO代表了一种共同的核信号，介导由这些不同的抗癌药物诱导的核-胞质信号通路。通过以下具体目的，我们将验证一系列NEMO翻译后修饰介导其核输入和输出，以伴随IKK调节因子从细胞核到细胞质，以促进IKK在基因毒性刺激下的激活。目的1：明确基因毒性物质修饰NEMO的SUMO-1机制。目的2：确定泛素在NEMO核输出基因毒性信号传导中的作用。目的3：阐明ATM在基因毒性物质激活IKK中的作用。拟议的研究将有助于确定NF-KB被某些基因毒性抗癌药物激活的基本机制。这些机制也可以作为其他基因毒性物质（如电离辐射）和其他靶向DNA拓扑异构酶I或II的物质诱导的激活途径的模型。此外，新的试剂工具，如磷酸化特异性NEMO抗体，编码野生型和突变型SUMO和NEMO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解剖NF-KB激活途径的努力有用。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/放射致敏。

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.