SH3结构域蛋白Dlish调控果蝇Hippo信号通路的分子机制研究

The giant Drosophila protocadherins Fat regulates proliferation via the growth-inhibiting Hippo pathway. These functions are shared by its mammalian homolog Fat4, and human mutation in Fat4 causes tumorigenesis. Despite its importance, only a little is known about how it regulates development and pathology. Using a combination of biochemistry and genetics screen, we have found two components of Fat signaling, App and newly discovered Dlish, this is the first time filled that physical gap, and in a way that provides a strong working model for the biochemistry of Fat signal transduction. Our data indicates that Fat inhibits the palmitoylation of Dlish through App and thereby its affinity for and accumulation near the subapical cell membrane. Our preliminary data indicate Dlish also directly binds Expanded and reduces its levels. We propose experiments designed to rigorously test and extend our understanding of this important, moving from in vitro to in vivo assays and analyses. The study may reveal how Dlish links between Fat signaling and Hippo signaling pathway. The results will give the theoretic basis and new idea for the study in the mammanial Hippo signaling pathway in the tumorigenesis.

果蝇巨大原钙粘蛋白Fat通过抑制生长的Hippo通路调控增殖。Fat在哺乳动物中的同源蛋白Fat4具有相似功能，人类Fat4突变导致肿瘤发生。尽管Fat信号很重要，但关于Fat怎样结合改变细胞行为而调控发育和疾病知之甚少。我们已经通过生化和遗传筛选，发现两个Fat信号成员App和新发现的Dlish，填补Fat信号连接上缺口，提出了Fat信号传导的重要生化模型。我们的数据表明Fat抑制App结合和棕榈酰化Dlish导致的细胞膜亚顶端的富集。前期实验我们发现Dlish直接结合Expanded且降低其蛋白水平，我们将设计严格的实验，采用体外到体内的实验方法来增加我们对这条重要信号通路的理解，鉴定通路中新的成员，揭示Fat信号如何连接Hippo信号通路，研究结果将为研究哺乳动物Hippo信号通路在肿瘤发生中的作用提供理论基础和新的思路。

本研究基于我们前期的实验中发现Dlish直接结合Expanded（Ex）且降低其蛋白水平，本研究设计严格的实验，采用体外到体内的实验方法来增加我们对这条重要信号通路的理解，揭示Fat信号如何连接Hippo信号通路。本研究通过免疫共沉淀结合质谱鉴定发现新的Fat信号响应蛋白Dlish与已知的Hippo信号通路上游调控蛋白Ex发生相互作用，Dlish与Ex蛋白C端多个SH3结构域结合。进一步的体内实验表明：Dlish降低细胞亚顶端的Ex蛋白水平，Dlish缺失将阻止由Fat缺失造成的Ex不稳定性。同时，Dlish与E3泛素连接酶Slimb结合，通过招募Slimb来泛素化Ex。研究结果将为研究哺乳动物Hippo信号通路在肿瘤发生中的作用提供理论基础和新的思路。