Regulation of Hippo and PCP signaling by the protocadherins Fat and Dachsous

项目摘要 巨大的果蝇原钙粘蛋白Fat和Dachsous（Ds）形成嗜异性的双向信号对 通过生长抑制Hippo途径调节增殖，平面细胞极性（PCP）通过 并且独立于“核心”PCP途径。这些功能由它们的哺乳动物同系物共享， 人类Fat和Ds突变导致Hennekam和货车的神经和多系统缺陷 Maldergem综合征。尽管它的重要性，只有一点是关于如何结合脂肪和Ds 改变细胞行为，从而调节发育和病理。 脂肪、Ds以及Hippo和PCP途径的效应物集中在细胞的亚顶端区域。 上皮细胞，脂肪的细胞内结构域（ICD）对两个细胞的亚顶端水平有很强的影响， 关键蛋白质第一种是支架肌球蛋白Dachs，它结合并抑制疣（Lats 1/2），最后一种是支架肌球蛋白Dachs。 Hippo途径中的效应激酶，并调节核心PCP途径中的Sple。二是 FERM支架蛋白扩展，刺激疣活动。然而，物理和生化 Fat ICD、Dachs和Expanded之间的联系从未建立。 通过结合蛋白质结合筛选、生物化学和遗传学，我们首次填补了 物理差距，并在某种程度上，提供了一个强大的工作模式的生物化学脂肪信号转导。 脂肪ICD结合两种蛋白质，DHHC棕榈酰转移酶近似（App）和新的 发现了SH 3衔接蛋白Dlish。我们的数据表明，脂肪通过抑制Dlish的棕榈酰化， 因此，它的亲和力和积累近根尖细胞膜。在脂肪突变体中， 膜相关Dlish增加，直接结合Dachs并将其募集到亚顶端细胞皮质， 达克斯抑制疣的地方Dlish也直接绑定扩展和减少其水平和疣刺激 活动，可能通过调节泛素化。我们提出了旨在严格测试和 扩展我们对这一重要而不寻常的信号通路的理解，从体外到体内 检测和分析，研究平行和替代途径，并确定新的途径 件.

Project Summary The giant Drosophila protocadherins Fat and Dachsous (Ds) form a heterophilic, bidirectional signaling pair that regulates proliferation via the growth-inhibiting Hippo pathway, and planar cell polarity (PCP) both through and independently of the “core” PCP pathway. These functions are shared by their mammalian homologs, and human mutations in Fat and Ds cause the neurological and multisystem defects of Hennekam and Van Maldergem syndromes. Despite its importance, only a little is known about how binding between Fat and Ds change cell behavior, and thus how it regulates development and pathology. Fat, Ds and the effectors of the Hippo and PCP pathways are concentrated in the subapical domain of epithelial cells, and the intracellular domain (ICD) of Fat has strong effects on the subapical levels of two critical proteins. The first is the scaffolding myosin Dachs, which binds and inhibits Warts (Lats1/2), the final effector kinase in the Hippo pathway, and which regulates Sple in the core PCP pathway. The second is the FERM scaffolding protein Expanded, which stimulates Warts activity. However, the physical and biochemical links between the Fat ICD, Dachs and Expanded have never been established. Using a combination of protein-binding screens, biochemistry and genetics, we have for the first time filled that physical gap, and in a way that provides a strong working model for the biochemistry of Fat signal transduction. The Fat ICD binds two proteins, the DHHC palmitoyltransferase Approximated (App), and the newly discovered SH3 adaptor protein Dlish. Our data indicates that Fat inhibits the palmitoylation of Dlish through App and thereby its affinity for and accumulation near the subapical cell membrane. In fat mutants, membrane-associated Dlish increases, directly binding Dachs and recruiting it to the subapical cell cortex, where Dachs inhibits Warts. Dlish also directly binds Expanded and reduces its levels and Warts-stimulating activity, likely through regulated ubiquitination. We propose experiments designed to rigorously test and extend our understanding of this important and unusual signaling pathway, moving from in vitro to in vivo assays and analyses, investigating parallel and alternative pathways, and identifying new pathway components.