TRAF6 E3 LIGASE FUNCTION IN OSTEOCLASTOGENESIS

描述(申请人提供)：RANK通过调节破骨细胞的分化和激活，在维持骨骼内稳中起核心作用。RANKL与其受体的结合导致TRAF6的募集，从而激活了NF-(B)、JNK、p38和MAPK通路。由于TRAF6缺陷小鼠发生严重的骨化病，TRAF6是关键的RAF6适配分子。最近的证据表明，TRAF6的N-末端环域是其信号传递所必需的，很可能是通过E3泛素连接酶，它与泛素结合酶复合体UBC13/Uev1A一起催化合成通过赖氨酸-63(K63)连接的独特的多泛素链。然而，这种独特的泛素修饰并不针对TRAF6的降解，而是TRAF6的自动泛素化作为支架来招募激活包括TAK1和IKK在内的激酶复合体所需的分子。 在这个应用中，我们提供了初步的数据来说明TRAF6自动泛素化在RANK信号中的功能作用。我们发现，在RANKL刺激下，内源性TRAF6通过K63连接被自动泛素化，这对NF-B的激活、NFATc1的诱导以及随后的破骨细胞分化是至关重要的。内源性TAB2和NEMO是潜在的TRAF6下游信号分子，在RANK激活后也被泛化。TRAF6的自动泛素化依赖于它的环区和UBC13，这两个蛋白质中的任何一个的突变都会扰乱RANK信号。引人注目的是，我们在TRAF6中发现了TRAF6自身泛素化所需的一个关键的Ub受体位点残基(K124)。该位点的突变使TRAF6介导的TAB2募集、TAB2和NEMO泛素化、TAK1和IKK激活、NF-B激活和破骨细胞分化消失。此外，我们提出的证据表明，TAK1的激酶活性是有效激活IKK和JNK、诱导NFATc1和RANKL诱导破骨细胞分化所必需的。通过结构作图研究，我们已经在TAK1的C末端确定了一个独特的结构域，它需要与TAB2及其同源物Tab3相互作用。由TAK1的最后100个残基组成的GFP融合蛋白的表达作为显性负突变，抑制RANKL信号转导和破骨细胞分化。这些结果支持一种新的依赖于Ub的TRAF6信号通路，这是有效的破骨细胞分化所必需的。 我们推测，在RANKL刺激破骨细胞前体细胞时，TRAF6通过其泛素连接酶活性来协调募集(通过TAB2和NEMO的位点特异性泛素化)和激活TAB2/TAK1和IKK复合体来激活NF-(B)途径，这是破骨细胞终末分化所必需的。为了解决这一假设，我们提出了三个综合的具体目标。1)确定RANKL治疗后原发骨髓中非蛋白酶体泛素化事件的特征。2)表征TRAF6自动泛素化在招募和泛化下游靶标方面的作用。3)建立和鉴定单核/巨噬细胞系中TAK1缺陷的条件性基因敲除小鼠，以确定TAK1在破骨细胞形成中的生理意义。总之，这些研究将揭示TRAF6通过泛素修饰调节破骨细胞分化的分子机制。特别是，从这些实验中收集的数据可以为开发各种与骨骼相关的疾病以及可能与免疫和炎症相关的其他疾病的治疗策略提供见解。

DESCRIPTION (provided by applicant): RANK plays a central role in the maintenance of the bone homeostasis by regulating the differentiation and activation of osteoclasts. The binding of RANKL to its receptor results in the recruitment of TRAF6, which activates the NF-(B, JNK, p38, and MAPK pathways. TRAF6 is the critical RANK adaptor molecule since TRAF6-deficient mice develop severe osteopetrosis. Recent evidence indicates that the N-terminal RING domain of TRAF6 is required for its ability to signal, most likely by functioning as an E3 ubiquitin ligase, which together with the ubiquitin conjugating enzyme complex Ubc13/Uev1A catalyzes the synthesis of a unique polyubiquitin chain linked through lysine-63 (K63). However, this unique ubiquitin modification does not target TRAF6 for degradation, but rather the auto-ubiquitination of TRAF6 serves as a scaffold to recruit molecules required for the activation of kinase complexes including TAK1 and IKK. In this application, we provide preliminary data to address the functional role of TRAF6 auto-ubiquitination in RANK signaling. We show that upon RANKL stimulation endogenous TRAF6 is auto-ubiquitinated via K63 linkages, which is critical for NF-(B activation, induction of NFATc1 and subsequent osteoclast differentiation. Endogenous TAB2 and NEMO, which are potential TRAF6 downstream signaling molecules, are also ubiquitinated upon RANK activation. TRAF6 auto-ubiquitination is dependent on its RING domain and Ubc13 and mutations in either of these proteins disrupt RANK signaling. Strikingly, we identified a single critical Ub acceptor site residue (K124) in TRAF6 that is required for TRAF6 auto-ubiquitination. Mutation of this site abolished TRAF6-mediated TAB2 recruitment, TAB2 and NEMO ubiquitination, TAK1 and IKK activation, NF-(B activation, and osteoclast differentiation. Furthermore, we present evidence that the kinase activity of TAK1 is required for the efficient activation of IKK and JNK, induction of NFATc1, and osteoclast differentiation by RANKL. Through structural mapping studies, we have identified a unique domain in the C-terminus of TAK1 that is required to interact with TAB2 and its homologue TAB3. Expression of a GFP-fusion protein consisting of the last 100 residues of TAK1 acts as a dominant negative mutant that suppresses RANKL signaling and osteoclast differentiation. These results support a novel Ub-dependent TRAF6 signaling pathway that is required for efficient osteoclast differentiation. We hypothesize that upon RANKL stimulation of osteoclast progenitors, TRAF6 functions through its ubiquitin ligase activity to coordinate the recruitment (via site-specific ubiquitination of TAB2 and NEMO) and activation of the TAB2/TAK1 and IKK complexes to activate the NF-(B pathway, which is required for terminal osteoclast differentiation. To address this hypothesis, we propose three integrated specific aims. 1) Characterize non-proteasomal ubiquitination events in primary BMM after RANKL treatment. 2) Characterize the role TRAF6 auto-ubiquitination for its ability to recruit and ubiquitinate downstream targets. 3) Determine the physiological significance of TAK1 in osteoclastogenesis by generating and characterizing conditional knock-out mice deficient in TAK1 in the monocyte/macrophage linage. Taken together, these studies will reveal the molecular mechanism by which TRAF6 through ubiquitin modification regulates osteoclast differentiation. In particular, the data gathered from these experiments may provide insights to developing therapeutic strategies for a variety of bone related disease and possibly other diseases associated with immunity and inflammation