Mechanisms for negative regulation ofTAK1/MAPKKK in innate immune responses

TAK 1是MAPKKK家族蛋白激酶的成员之一，在白细胞介素1信号通路和Toll样受体信号通路中起着关键作用。在配体结合后，TAK 1被快速且瞬时地激活。尽管对TAK 1的激活机制已有广泛的研究，但TAK 1的激活是如何终止的仍不清楚。为了鉴定TAK 1的负调节因子，我们搜索了与活化的TAK 1形成复合物的蛋白质。为此，我们首先建立了HeLa细胞稳定表达串联亲和纯化（TAP）标记的TAK 1在接近内源性的水平。然后，从IL-1 β刺激的细胞中纯化TAP-TAK 1，并通过质谱鉴定共纯化蛋白。我们鉴定了一种E3泛素连接酶X作为一种新的TAK 1结合蛋白。生化分析表明，X可以强烈抑制TAK 1诱导的NF-κB活化，X的共表达显著降低TAK 1活化剂TAB 1的量。这些结果表明，X是一个很好的候选人尚未被确定的TAK 1的负调节。本研究将有助于我们全面了解TAK 1在细胞因子信号传导和先天免疫应答中的调节作用，并研究了HTLV-1 Tax蛋白激活TAK 1的机制。我们发现Tax与衔接蛋白TAB 2相互作用并激活TAK 1。税收的普遍化可能是其激活TAK 1能力的关键。重要的是，我们证明TRAF家族蛋白中的TRAF 2、5和6可以诱导Tax的多聚泛素化。据我们所知，这是第一个关于细胞E3泛素连接酶对Tax的报道。这项研究将有助于阐明Tax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.