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

项目摘要 巨大的果蝇原钙粘附素Fat和Dachsous(D)形成了一对异嗜性的双向信号对 它通过抑制生长的河马途径和平面细胞极性(PCP)来调节增殖，两者都是通过 并且不依赖于“核心”PCP途径。这些功能是由它们的哺乳动物同源物共享的，并且 人类脂肪和D基因突变导致Hennekam和Van的神经系统和多系统缺陷 马尔德吉姆综合征。尽管它很重要，但人们对脂肪和脂肪之间的结合知之甚少 改变细胞的行为，从而改变其调节发育和病理的方式。 脂肪、D和河马和PCP通路的效应器集中在 上皮细胞，而脂肪的胞内区(ICD)对两个细胞的亚尖水平有很强的影响 关键蛋白质。第一种是支架肌球蛋白达克斯，它结合并抑制疣(Lats1/2)，最后一种是 河马途径中的效应蛋白激酶，并调节核心PCP途径中的SPLe。第二个是 FERM支架蛋白膨胀，刺激疣活动。然而，生理和生化 Fat ICD、Dachs和Expanded之间的联系从未建立过。 利用蛋白质结合筛选、生物化学和遗传学的组合，我们第一次填满了 物理差距，并以一种方式提供了一个强有力的工作模型的生物化学的脂肪信号转导。 Fat ICD结合两种蛋白质，DHHC棕榈酰转移酶近似(App)和新的 发现了SH3接头蛋白DLISH。我们的数据表明，脂肪通过抑制DLISH的棕榈酰化 APP及其与亚根尖细胞膜的亲和力和在亚尖细胞膜附近的积累。在脂肪突变体中， 膜相关的DLISH增加，直接结合DACH并将其招募到心尖下细胞皮质， 达克斯抑制疣的地方。DLISH还直接绑定扩展和减少其水平和尖锐湿疣刺激 活动，可能是通过调节的泛素化。我们提出的实验旨在严格测试和 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 分析和分析，调查平行和替代途径，并确定新途径 组件。

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.