Hippo信号通路新的上游调控激酶筛选及调控机制研究

The FERM-domain protein Expanded (Ex) is one of the upstream regulators that are critical to activation of the Hippo signaling. Our previous study indicates that the Drosophila E3 ubiquitin ligase SCF[slmb] mediates the degradation of Ex through the ubiquitination-proteasome pathway. SCF[slmb] normally recognizes and binds to specific motifs (phospho-degron, which is often phosphorylated prior to its recognition by SCF[slmb]) on its substrates, however, the specific kinase(s) regulating Ex phosphorylation and degradation remains yet to be identified. Crumbs (Crb) has been shown to interact with Ex, leading to Ex phosphorylation on multiple sites and subsequent degradation. Our own work also show that Wts, a core kinase in Hippo pathway, promote Ex protein stability. These studies suggest a important regulatory role of protein kinases in modulating Ex function. We will perform screens in Drosophila to identify novel kinases involved in the Hippo pathway and, in particular, investigate the mechanisms of these kinases in the regulation of Ex stability by employing combinatorial techniques including genetics, cellular and molecular biology and biochemistry. The study proposed here is of critical significance both to elaborate mechanisms that fine-tune the evolutionarily conserved Hippo signaling, and to enhance our knowledge about the etiology of human diseases such as tumorigenesis that were found to be associated with Hippo pathway dysregulation.

Expanded（Ex）蛋白是Hippo信号通路重要的上游调控因子之一。我们前期的研究发现果蝇中SCF[slmb]泛素连接酶直接负责Ex蛋白的泛素化降解。SCF[slmb]通常结合到底物中磷酸化降解决定元（phospho-degron）上，但目前负责这一磷酸化的激酶尚未发现。Crumbs与Ex的结合引起Ex多磷酸化并促进其降解；我们还发现Hippo通路核心激酶Wts具有稳定Ex蛋白的功能。这些结果提示我们激酶在调控Ex蛋白活性中起到至关重要的作用。该申请计划对果蝇基因组中的所有激酶进行筛选，通过遗传学、细胞生物学及生物化学等方法揭示包括Wts在内的激酶如何调控Ex稳定性的机制。这一研究对于深入阐明Hippo通路如何精细调控细胞增殖的作用机制机制具有重要意义。同时，鉴于Hippo通路的高度保守性，该研究也可能为揭示因Hippo通路失调引起的肿瘤发生机理提供重要线索。

Expanded（Ex）蛋白是Hippo信号通路重要的上游调控因子之一。我们前期的研究发现果蝇中SCF[slmb]泛素连接酶直接负责Ex蛋白的泛素化降解。SCF[slmb]通常结合到底物中磷酸化降解决定元（phospho-degron）上，但目前负责这一磷酸化的激酶尚未发现。Crumbs与Ex的结合引起Ex多磷酸化并促进其降解；我们还发现Hippo通路核心激酶Wts具有稳定Ex蛋白的功能。这些结果提示我们激酶在调控Ex蛋白活性中起到至关重要的作用。在该项目执行过程中，我们通过遗传筛选鉴定了Hippo通路新的上游调控因子Shaggy（Sgg）激酶；发现通路上游调控信号，揭示一种新型通路互作机制（Wg-Hippo）调控组织生长发育。通过遗传和免疫学方法，基本确定了上述调控信号作用于Hippo通路的分子节点和工作模型，为进一步深入挖掘机理、拓展Hippo通路新的调控机制（如组织特异性调控机制）打下了坚实基础。本项目的开展也为将来在蛋白质翻译后修饰（磷酸化，泛素化等修饰组学）对Hippo通路的调控、Hippo信号交叉调控等领域积累丰富资本。我们目前正在解析Hippo-Wnt互作在干细胞增殖和稳态中的功能（延伸研究）。通过该项目的实施，我们一共发表包括Developmental Cell在内的研究论文5篇。培养博士研究生4名、硕士研究生1名。