Hippo信号传导通路Lats1/2激酶的底物筛选及功能研究

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。

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.