Mechanisms for negative regulation ofTAK1/MAPKKK in innate immune responses

TAK1是MAPKKK家族中的一员，在白介素1信号通路和Toll样受体信号通路中发挥着重要作用。在配体结合后，TAK1被快速和瞬时激活。虽然对TAK1的激活机制已经有了广泛的研究，但目前仍不清楚TAK1的激活是如何终止的。为了确定TAK1的负调控因子(S)，我们搜索了与激活的TAK1形成复合体的蛋白质(S)。为此，我们首先建立了稳定表达串联亲和纯化(TAP)标记的TAK1的HeLa细胞，其表达水平接近内源性TAK1。然后，从IL-1β刺激的细胞中纯化TAP-TAK1，并用质谱仪鉴定共纯化蛋白。我们发现E3泛素连接酶X是一种新的TAK1结合蛋白。生化分析表明，X能强烈抑制Tak1诱导的NF-κB的激活，X的共表达显著降低Tak1-激活剂TAB1的含量。这些结果表明，X是一个很好的候选的TAK1的负性调控因子。这项研究将有助于我们全面了解TAK1在细胞因子信号转导和先天免疫反应中的调控机制。我们还研究了HTLV-1 Tax蛋白激活TAK1的机制。我们发现，TAX与一种接头蛋白TAB2相互作用，激活TAK1。税收的泛素化可能对其激活TAK1的能力至关重要。重要的是，我们证明了TRAF家族蛋白中的TRAF2、TRAF5和TRAF6可以诱导Tax的多泛素化。据我们所知，这是关于细胞内E3泛素连接酶与税收的首次报道。这项研究将有助于阐明税收致癌作用的分子机制。

TAK1, a member of the MAPKKK family protein kinases, plays a pivotal role in interleukin-1 signaling pathway as well as Toll-like receptor signaling pathway. Upon ligand binding, TAK1 is rapidly and transiently activated. Although there have been extensive studies on the activation mechanism of TAK1, it is still unclear how TAK1 activation is terminated. To identify negative regulator(s) for TAK1, we searched protein(s) that form a complex with activated TAK1. To this end,we first established HeLa cells stably expressing Tandem affinity purification (TAP)-tagged TAK1 at the level close to the endogenous one. Then, TAP-TAK1 was purified from the cells stimulated with IL-1beta, and co-purifiying proteins were identified by mass spectrometry. We identified an E3 ubiquitinligase X as a novel TAK1-binding protein. Biochemical analysis revealed that X can strongly inhibit TAK1-induced activation of NF-κB and coexpression of X significantly reduces the amount of TAK1-activator, TAB1. These results suggest that X is a good candidate for yet-to-be-identified negative regulator of TAK1. This study will contribute to our full understanding of how TAK1 is regulated in cytokine signaling as well as innate immune responses.We have also studied a mechanism of TAK1 activation by HTLV-1 Tax protein. We found that Tax interacts with an adapter protein TAB2 and activates TAK1. Ubiquitination of Tax is likely to be essential for its ability to activate TAK1. Importantly,we demonstrated that TRAF2, 5, and 6 ofTRAF family proteins can induce polyubiquitination of Tax. To our knowledge, this is the first report on the cellular E3 ubiquitinligases towards Tax. This study should shed light on the molecular mechanisms for the oncogenic action of Tax.