蛋白激酶MAP3K8激活后降解的生化机制

The protein kinase MAPKKK family member MAP3K8 is important for activation of MEK1/2-ERK1/2 pathway in the Toll-like Recptors(TLRs), IL1R, TNFR signaling pathways and is required for induction of proinflammatory cytokines especially the production of TNFa. It is also an oncogene and overexpressed in many tumors. MAP3K8 forms stable complex with NF-kB1(p105) and ABIN2 in resting cells, with the latter two subunits regulating the cellular steady-state level of MAP3K8.When bound to their cognate ligands, TLRs and IL1R recruit RING-finger-containing E3 ubiquitin ligase TRAF6, which, together with heterodimeric E2 ubiquitin conjugating enzyme, catalyze synthesis of unanchored K63-linked polyubiquitin chains. These unanchored, free polyubiquitin chains in turn lead to activation of TAK1 kinase complex and subsequently IKK kinase complex.Activated IKK in turn phosphorylates p105 and leads to its degradation by the ubiquitin-proteasome system. Degradation of p105 releases MAP3K8 leading to its activation in an unknowm mechanism. Following activation, MAP3K8 is quickly degraded, a process called activation-induced degradation. Although MAP3K8 is important in innate immune and proinflammatory responses, the biochemical mechanism of its activation and subsequent degradation is not understood yet. We have reconsitituted the activation and degradation of MAP3K8 in a cell-free system by using recombinant TRAF6 protein as the activator, which recapitulates the activation and degradation of MAP3K8 in vivo when cells are stimulated by IL1b or LPS. With this system, we are addressing the activation mechanism of MAP3K8, a project that is being funded by NSFC. In this proposal, we are trying to investigate the biochemical mechanism of MAP3K8 degradation following its activation and the pathophysiological significance of activation-induced MAP3K8 degradation.

丝氨酸／苏氨酸蛋白激酶MAPKKK家族成员MAP3K8在参与固有免疫和炎症反应的细胞如巨嗜细胞以及甲状腺癌、乳腺癌中表达，介导TNFR,IL1R和Toll-like Receptor(TLR)等信号通路中MEK1/2-ERK1/2的激活，从而调节促炎症因子特别是TNFa的表达。当这些信号通路激活后，MAP3K8首先被激活然后被快速降解。然而，在这些信号通路中MAP3K8的激活以及激活后降解的生化机制知之甚少、有待深入研究。我们在体外无细胞系统中重建了MAP3K8的激活和降解过程。利用该系统，在上一个国家自然科学基金项目（在研）中，通过经典液相色谱蛋白分离技术，我们分离、纯化和鉴定了MAP3K8激活所必需的分子和条件，探讨了其激活的生化机制。作为延续，在本申请中，我们拟利用该系统和现有的优势和积累的材料来深入研究MAP3K8激活后被调控性降解的机制。

丝氨酸／苏氨酸蛋白激酶MAPKKK家族的MAP3K8在参与固有免疫和炎症反应的细胞比如巨嗜细胞以及甲状腺癌以及乳腺癌中表达，介导TNFR,IL1R以及Toll-like Receptor(TLR)等信号通路中MEK1/2-ERK1/2的激活，从而调节促炎症因子（pro-inflammatory cytokines）特别是TNF的表达。当这些信号通路激活后，MAP3K8首先被激活然后被快速降解。发生类似行为的蛋白激酶是不多的。然而，在这些信号通路中MAP3K8的激活以及激活后快速降解的生化机制还有待深入研究。围绕MAP3K8，本课题拟回答如下关键问题：1）介导 MAP3K8 诱导性降解的转译后修饰形式和位点以及泛素化位点的鉴定；2）介导 MAP3K8 泛素化的泛素连接酶 SCF 复合体的鉴定；3）MAP3K8 激活后快速降解的生理和病理意义。我们的研究结果表明：a）MAP3K8在激活过程中，它的Ser60、Ser62、Ser66、Ser141、Tyr153、Ser413、Ser443发生了高度磷酸化；b）基于Cul1的SCF泛素连接酶复合物介导了MAP3K8的降解，而F-box蛋白FBXO3是识别磷酸化后的MAP3K8的接头蛋白。另外，通过小分子抑制剂文库的筛选，我们得到了6个MAP3K8的抑制剂，这些可以作为潜在的先导药物，进行进一步的改造，用以提高其特异性抑制活性。我们的研究鉴定了介导MAP3K8激活后发生降解的泛素连接酶，加深了对MAP3K8功能调控、以及其生理病理意义的了解。在该课题资助下，我们已经发表了3篇高水平论文，在国际国内学术会议上报告5次。