Mechanisms of Protein Kinase Activation by Ubiquitin in the NF-kB Pathways

描述（由申请人提供）：转录因子NF-kB在许多生理和病理过程中起关键作用，包括免疫和炎症反应。NF-kB的活性主要通过其抑制剂IkB的磷酸化和降解受到严格调控。通过泛素-蛋白酶体途径降解IkB，使NF-kB进入细胞核调节基因表达。有趣的是，IKK激酶对IkB的磷酸化是由k63连接的多泛素化通过蛋白酶体不依赖的机制调节的。我们之前的研究表明，IKK的激活需要由Ubc13和Uev1A组成的泛素结合酶复合物、泛素连接酶TRAF6和上游蛋白激酶TAK1复合物。TAK1复合体含有接头蛋白TAB2和TAB3，它们具有能够结合K63多泛素链的特殊泛素结合结构域。多泛素链与TAK1复合物的结合导致该激酶的激活，进而磷酸化并激活IKK。IKK的激活需要它的调控亚基NEMO，它包含两个泛素结合结构域。NEMO及其泛素结合在IKK激活中的作用尚不完全清楚。我们最近的研究表明，K63多泛素化对于白细胞介素- 1激活IKK至关重要。（IL-1?），它激活TRAF6。此外，我们发现TRAF6和Ubc13/Uev1A催化非锚定多泛素链的合成，直接激活TAK1激酶复合物。有趣的是，IKK被肿瘤坏死因子-？（TNF?）需要Ubc5和cIAPs分别作为E2和E3使RIP1多泛素化，但不需要形成K63多泛素链。这些结果表明泛素调控蛋白激酶的复杂性出乎意料。在这个应用中，我们建议阐明在IL-1中TAK1和IKK激活的独特泛素依赖机制。（Aim 1）和TNF？（目标2）途径。此外，我们将研究NEMO结合多泛素调控IKK激活的机制（Aim 3）。

DESCRIPTION (provided by applicant): The transcription factor NF-kB plays a pivotal role in many physiological and pathological processes, including immune and inflammatory responses. The activity of NF-kB is tightly regulated, primarily through phosphorylation and degradation of its inhibitors, IkB. The degradation of IkB by the ubiquitin- proteasome pathway allows NF-kB to enter the nucleus to regulate gene expression. Interestingly, phosphorylation of IkB by the kinase IKK is regulated by K63-linked polyubiquitination through a proteasome-independent mechanism. Our previous studies have shown that IKK activation requires the ubiquitin-conjugating enzyme complex consisting of Ubc13 and Uev1A, the ubiquitin ligase TRAF6, and the upstream protein kinase TAK1 complex. The TAK1 complex contains the adaptor proteins TAB2 and TAB3, which harbor specialized ubiquitin-binding domains capable of binding to K63 polyubiquitin chains. The binding of polyubiquitin chains to the TAK1 complex leads to the activation of this kinase, which in turn phosphorylates and activates IKK. IKK activation requires its regulatory subunit NEMO, which contains two ubiquitin-binding domains. The role of NEMO and its ubiquitin-binding in IKK activation is not fully understood. Our recent studies demonstrate that K63 polyubiquitination is essential for IKK activation by interleukin- 1? (IL-1?), which activates TRAF6. Moreover, we have found that TRAF6 and Ubc13/Uev1A catalyze the synthesis of unanchored polyubiquitin chains, which directly activate the TAK1 kinase complex. Interestingly, IKK activation by tumor necrosis factor-? (TNF?) requires polyubiquitination of RIP1 by Ubc5 and cIAPs as the E2 and E3, respectively, but does not require the formation of K63 polyubiquitin chains. These results suggest an unanticipated complexity of protein kinase regulation by ubiquitin. In this application, we propose to elucidate the distinct ubiquitin-dependent mechanisms of TAK1 and IKK activation in the IL-1? (Aim 1) and TNF? (Aim 2) pathways, respectively. In addition, we will investigate the mechanisms by which polyubiquitin binding by NEMO regulates IKK activation (Aim 3).