Hippo信号传导通路Lats1/2激酶的底物筛选及功能研究

The homeostasis of organ size is one of the most important features of multi-cellular organisms. However, the molecular mechanism of organ size regulation is a long-standing mystery. Recently, a new signaling pathway called the Hippo pathway was established to play important roles in organ size regulation and tumorigenesis. Lats1/2 are key kinases in the Hippo pathway, which phosphorylate and inhibit YAP transcription co-activator to inhibit proliferation and promote apoptosis. Furthermore, there is plenty of evidence supporting many other cellular functions of Lats1/2, such as cell differentiation, cytokinesis, senescence, autophagy, centrosome duplication, and neuron dendritic tiling. However, unlike this diverse set of functions, there is only very limited knowledge on the substrates of Lats1/2. Dyrk1/2 are the only known direct Lats1/2 phosphorylation substrates besides YAP and its paralog TAZ. In fact, many of the Lats1/2 functions could not be explained by known substrates. Therefore, it is important to systematically identify all Lats1/2 substrates encoded by the genome in order to understand the biological functions of Lats1/2 and the Hippo pathway. In this project, we proposed two complementary methods to screen for Lats1/2 substrates. First, we use the newly developed chemical genetic approach for kinase substrate mapping. And second, we use Phos-tag gel shift assay to screen Lats1/2 target consensus motif-containing proteins for Lats1/2 substrates. We would then further study the molecular mechanisms of phosphorylation in regulation of the functions of 1-2 important substrates. Moreover, we would analyze the role of the Hippo pathway in the biological functions executed by these substrates. Completion of this project will give us a clear picture of the substrate spectrum of Lats1/2 and significantly broaden the biological functions of Lats1/2 and the Hippo pathway.

Hippo通路是一条调控多细胞生物器官大小的磷酸化信号传导通路。Lats1/2是Hippo通路中将磷酸化信号传递给效应分子的关键激酶。它磷酸化并抑制YAP转录辅激活因子从而调控细胞增殖和凋亡。然而除YAP之外Lats1/2的底物研究很少。另一方面， Lats1/2具有许多其它功能，例如调控细胞分化、老化、自噬、中心体复制、神经元树突覆盖性等。介导这些功能的底物尚不明确。因此全面鉴定Lats1/2的底物是阐明Lats1/2功能的迫切需要。本课题拟结合化学遗传激酶底物筛选法和基于磷酸化位点基序的Phos-tag凝胶电泳迁移法在全基因组水平筛选Lats1/2激酶的底物，并以1-2个重要底物为例阐明Lats1/2通过磷酸化调控这些蛋白质并发挥生物学功能的分子机制。本课题的完成将描绘Lats1/2的底物谱，拓宽Lats1/2及Hippo通路的研究范围，提升对Hippo通路生物学意义和应用价值的认识。

Hippo信号转导通路在器官大小调控、癌症发生、组织再生、以及干细胞的功能上发挥重要作用。该通路的核心部分是一个由Mst1/2和Lats1/2蛋白激酶组成的激酶链组成。激酶的生物学功能主要由底物决定，然而，除经典的YAP/TAZ转录辅激活因子以外，目前对Lats1/2下游底物的了解还非常少。本研究发现Lats1/2激酶能够磷酸化angiomotin（AMOT）的HXRXXS保守基序。磷酸化后能够抑制AMOT与F-actin的直接结合，进而影响F-actin应力纤维与粘着斑的形成。进一步研究发现AMOT磷酸化以后，能够抑制血管内皮细胞的迁移，这与细胞骨架及细胞粘着在细胞迁移中的重要作用相吻合。发育过程中的血管生成依赖于细胞的迁移。体内研究表明AMOT磷酸化抑制斑马鱼的发育过程中的血管生成。这一发现，鉴定出Hippo通路中新的下游底物AMOT，并且第一次报道了Hippo通路通过磷酸化AMOT调节血管的生成，扩展了对Hippo抑癌通路生理功能的认识。.负反馈调控是信号转导的一条普遍法则，它保证某种反应只响应达到阈值的真实信号，而对背景噪音信号不敏感。Hippo信号通路作为具有很强促生长能力的信号通路它的活性必须受到严格调控以避免肿瘤等过度生长性疾病。然而，这样的机制在哺乳动物的Hippo信号通路中以前并未发现。我们在研究中意外地发现在表达激活型Hippo通路效应分子YAP的细胞中，内源YAP发生很强的磷酸化，表明其被抑制。进一步研究发现YAP通过与TEAD转录因子形成复合物直接结合Lats2和AMOTL2的启动子，进而激活这两个Hippo信号通路基因的表达，从而形成抑制YAP活性的负反馈回路。我们发现这一负反馈机制在细胞接触和血清等生理刺激的作用下以及在小鼠肝脏内都存在，表明我们发现的这一机制对维持环境刺激下Hippo通路活性的稳态，阻止肿瘤发生具有重要的作用。