蛋白激酶TAK1激活的生化机制研究

Protein kinase TAK1, also known as MAP3K7 and MEKK7, is a very important signaling protein involved in the regulation of innate an adaptive immune responses, neural fold morphogenesis, vascular development, and tumorogenesis. Our previous studies have shown that in the TLR/IL-1R signaling pathways, the ubiquitin ligase E3 TRAF6catalyzes the synthesis of Lys63-linked free polyubiquitin chains. These free polyubiquitin chains then bind to TAB2 (or TAB3) and mediate dimerization or oligomerization of TAK1 complexes resulting in auto-phosphorylation of T184 and T187 in the kinase domain of TAK1 and subsequent activation of TAK1. Numerous studies indicate that T184/T187 phosphorylation is very essential for TAK1 activation. However, many studies also showed that Thr184/Thr187 phosphorylation is not sufficient for TAK1 activation, implying more regulations such as other post-translational modifications are required for TAK1 activation. We aim to explore other requirement for TAK1 activation. Our recent mass spectrometry-based studies have identified Ser412 in the C-terminus of TAK1 being heavily phosphorylated upon TLR/IL-1R activation, implying its involvement in TAK1 activation. In this proposal, we aim to study the importance of Ser412 phosphorylation for TAK1 activation, explore its relationship with Thr187 phosphorylation, identify kinase(s) that mediates Ser412 phosphorylation, and investigate the regulation of Ser412 phosphorylation and the kinase(s) in the TLR/IL-1R signaling pathways.

蛋白激酶 TAK1，在天然免疫、获得性免疫、TLR/IL-1R 等信号转导通路以及神经褶、血管发育、肿瘤发生中起着很重要的作用。我们前期的研究表明在TLR/IL-1R信号通路中，泛素连接酶TRAF6催化合成的K63链接的自由多泛素链与TAB2结合，引起TAK1激酶复合体的寡聚化，从而引起TAK1的T184和T187位点发生自磷酸化，导致TAK1的激活。然而有数据表明TAK1激酶结构域中的T184和T187位点的自磷酸化是TAK1激活的必要但不充分条件。在本申请中，我们拟探索TAK1激活所必需的其他磷酸化位点。利用质谱分析我们鉴定到TAK1在TLR/IL-1R 激活情况下，C端S412位点在有很强的磷酸化信号。据此，我们假定S412位点磷酸化是TAK1激活所必需的。我们将研究S412磷酸化对TAK1激活的重要性，在此基础上，进一步研究S412磷酸化位点以及该位点蛋白激酶的调控及其生理意义。

转化生长因子-β激活激酶1（TAK1）是NF-κB信号通路（如Toll样受体和白介素1受体信号）激活过程中的关键激酶。尽管现有证据表明TAK1的激活需要位于其活化环的第184位和第187位苏氨酸的磷酸化，但人们对TAK1完全激活的分子机制仍缺乏深入了解。本研究发现TAK1的C末端的卷曲螺旋结构域所介导的TAK1蛋白二聚化和自身磷酸化调控了第187位苏氨酸的磷酸化。更重要的是，在多种炎症信号如TNF-α、LPS和IL-1β刺激下，TAK1的完全激活还需要其第412位丝氨酸的磷酸化。体外的激酶实验和在体内利用shRNA干扰内源基因表达的方法发现cAMP依赖的蛋白激酶催化亚基α（PKACα）和X连锁蛋白激酶（PRKX）催化了该位点的磷酸化，并参与调节TLR/IL-1R信号的激活和下游炎性细胞因子的产生。利用Morpholino干扰体内基因表达和回补突变体实验证实了斑马鱼的TAK1蛋白上相对应的第391位丝氨酸位点也在NF-κB激活过程中发挥重要作用。本研究揭示出了PKACα和PRKX通过磷酸化调节TAK1激酶活力来调控天然免疫信号的新的分子机制。