Ubiquitin-Mediated Activation of the IkB Kinase Complex

描述（由申请人提供）：转录因子NF-κ B是 主要通过与抑制蛋白的结合来调节 伊卡帕NF-κ B/IkappaB复合物通常被隔离在细胞质中 直到细胞用激动剂如白细胞介素-1（IL-1）刺激，肿瘤 坏死因子（TNF）和脂多糖（LPS）。刺激时 在细胞中，IkappaB被IkappaB激酶复合物（IKK）快速磷酸化， 然后通过泛素-蛋白酶体途径降解。1 kappaB的降解 允许NF-κ B进入细胞核，打开下游基因，许多 在细胞的生与死中起着关键作用。 NF-κ B信号通路的关键步骤是IkappaB的磷酸化 被IKK综合体这种激酶复合物整合了来自多个 途径，包括从IL-1和LPS发出的途径。遗传学研究 证明TRAF 6，一种RING指结构域蛋白，和TRAF 6的组分， IKK复合物对IL-1和LPS激活NF-κ B是必需的。 然而，目前尚不清楚TRAF 6如何激活IKK。最近的研究， 主要研究者的实验室已经表明，IKK的激活 TRAF 6的复合物需要二聚体泛素缀合酶复合物， Ubc 13/Uev 1A，并形成一个独特的多聚泛素链连接，通过 泛素的赖氨酸-63（K63）。本提案的目的是了解 IKK复合物的泛素依赖性激活的新机制。 具体而言，实验提出1）研究结构和功能 Ubcl 3/Uev 1A和TRAF 6与IKK激活的关系; 2）鉴定和 表征TKAF 6激活IKK所需的其他因素，包括 泛素化靶点; 3）鉴定和表征K63连接的 多聚泛素链结合蛋白; 4）探讨其作用机制 TRAF 6对IKK的泛素依赖性激活。综合来看，这些研究 应该填补NF-κ B信号通路的重要空白，并提供 解决IKB难题所需的重要部分 通过泛素激活激酶。考虑到NF-κ B在人类中的重要性， 疾病，从拟议的研究中获得的信息将直接 与生物医学相关，包括发现新的治疗靶点。

DESCRIPTION (provided by applicant): The transcription factor NF-kappaB is regulated primarily through its association with the inhibitory protein IkappaB. The NF-kappaB/IkappaB complex is normally sequestered in the cytoplasm until cells are stimulated with an agonist such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and lipopolysaccharides (LPS). Upon stimulation of cells, IkappaB is rapidly phosphorylated by an IkappaB kinase complex (IKK) and then degraded by the ubiquitin-proteasome pathway. The degradation of 1kappaB allows NF-kappaB to enter the nucleus to turn on downstream genes, many of which play pivotal roles in the life and death of cells. A key step in the NF-kappaB signaling pathway is the phosphorylation of IkappaB by the IKK complex. This kinase complex integrates signals from multiple pathways including those emanating from IL-1 and LPS. Genetics studies have demonstrated that TRAF6, a RING finger domain protein, and components of the IKK complex are essential for NF-kappaB activation in response to IL-1 and LPS. However, it is not known how TRAF6 activates IKK. Recent studies by the principal investigator's laboratory have shown that the activation of IKK complex by TRAF6 requires a dimeric ubiquitin conjugating enzyme complex, Ubc13/Uev1A, and the formation of a unique polyubiquitin chain linked through lysine-63 (K63) of ubiquitin. The goal of this proposal is to understand the novel mechanisms of ubiquitin-dependent activation of the IKK complex. Specifically, experiments are proposed to 1) study the structure and function of Ubcl3/Uev1A and TRAF6 as related to IKK activation; 2) identify and characterize additional factors required for IKK activation by TKAF6, including the ubiquitination target; 3) identify and characterize the K63-linked polyubiquitin chain binding protein; 4) investigate the mechanisms of ubiquitin-dependent activation of IKK by TRAF6. Taken together, these studies should fill significant gaps in the NF-kappaB signaling pathway, and provide the important pieces that together are required to solve the puzzle of IKB kinase activation by ubiquitin. Given the importance of NF-kappaB in human diseases, information gained from the proposed studies will be of direct relevance to biomedicine, including discovery of novel therapeutic targets.