Mechanisms for negative regulation ofTAK1/MAPKKK in innate immune responses

TAK1是MAPKKK家族蛋白激酶的一员，在白细胞介素-1信号通路和toll样受体信号通路中起关键作用。在配体结合后，TAK1被迅速而短暂地激活。虽然对TAK1的激活机制已经有了广泛的研究，但TAK1的激活是如何终止的仍不清楚。为了鉴定TAK1的负调节因子，我们寻找了与活化TAK1形成复合体的蛋白。为此，我们首先建立了在接近内源性TAK1的水平上稳定表达串联亲和纯化（TAP）标记的TAK1的HeLa细胞。然后，从il -1 - β刺激的细胞中纯化TAP-TAK1，并用质谱法鉴定共纯化蛋白。我们发现E3泛素连接酶X是一种新的tak1结合蛋白。生化分析显示，X能强烈抑制tak1诱导的NF-κB活化，X的共表达可显著降低tak1激活因子TAB1的量。这些结果表明，X是尚未确定的TAK1负调节因子的良好候选者。这项研究将有助于我们全面了解TAK1如何在细胞因子信号传导和先天免疫反应中受到调节。我们还研究了HTLV-1 Tax蛋白激活TAK1的机制。我们发现，Tax与一个适配器蛋白TAB2相互作用，并激活TAK1。Tax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TRAF2、5和6的traff家族蛋白可以诱导Tax的多泛素化。据我们所知，这是第一个关于细胞E3泛素连接酶的报道。本研究将进一步揭示其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.