Regulation of IKK-epsilon and TRAF6 functions by posttranslational modifications

革兰氏阴性菌表面的脂多糖会触发复杂的细胞内信号级联反应，激活转录因子，如转录因子，从而诱导免疫调节基因的表达。在过去的几年里，已经发现了内毒素诱导的细胞内信号系统的许多不同的组成部分，包括蛋白激酶IKK和泛素E3连接酶TRAF6。由于对它们活动的控制还不是很清楚，本项目旨在通过翻译后修饰来研究它们之间的相互调节。初步结果显示，IKKα在3个丝氨酸上磷酸化TRAF6，而TRAF6在9个赖氨酸上介导诱导的泛素化。为了解决这些翻译后修饰的功能作用，敲除细胞将被重组，以稳定地表达这些在修饰氨基酸中发生变化的野生型和突变形式的蛋白质。这些细胞将通过各种实验方法进行分析，包括细胞生物学和生化实验。这些修饰的发生、动力学和调节是由修饰特异性抗体追踪的。随着最近的证据表明ikk在细胞核中的作用，CHIP-SEQ实验将揭示该激酶的基因组结合位点的全面图谱。这一基础研究项目将拓宽我们对促炎信号转导中发生的分子过程的理解，因此具有生物医学意义。

Lipopolysaccaride (LPS) contained on the surface of gram-negative bacteria triggers complex intracellular signaling cascades activating transcription factors such as NF-ýB that serve to induce the expression of immune regulatory genes. The last years have witnessed the identification of many different components of the LPS-inducible intracellular signaling system, including the protein kinase IKKý and the ubiquitin E3 ligase TRAF6. As the control of their activity is not well known, this project aims to study their mutual regulation by posttranslational modifications. Preliminary results show that IKKý phosphorylates TRAF6 at 3 serines, while TRAF6 mediates inducible ubiquitination of IKKý at 9 lysines. To address the functional role of these posttranslational modifications, knock-out cells will be reconstituted to stably express these proteins in their wildtype and mutated forms that are changed in the modified amino acids. These cells will be analyzed by a large variety of experimental approaches including cell biological and biochemical experiments. The occurrence, kinetics and regulation of these modifications are traced by modification-specific antibodies. As recent evidence shows a role of IKKý in the nucleus, ChIP-Seq experiments will reveal a comprehensive map of genomic binding sites for this kinase. This basic research project will broaden our understanding of molecular processes that occur in proinflammatory signal transduction and are thus of biomedical interest.