Control and Function of Ndr/LATS Signaling Systems

描述（由申请人提供）：GCK组“Mst/hippo”激酶调节AGC组“Ndr/LATS”激酶的信号通路是真核细胞生长、增殖和结构的古老控制器。我们的广泛目标是定义这些途径调节的不同细胞内过程，并确定这种控制的机制。Mst/hippo信号（“河马疣”途径）通过抑制YAP/ yorkiy相关转录共激活因子抑制后生动物细胞增殖的形式已成为最近关注的主题。然而，该途径独特且高度保守的“河马-trc”形式，其中大的“毛茸茸”相关蛋白介导Mst/河马激酶活化的Ndr/三角角激酶具有显着不同的功能；相对而言，我们对它们知之甚少，它们是本项目的重点。海马-trc通路对细胞伸展的极化生长和组织、神经元形态发生、有丝分裂纺锤体组织和细胞增殖的积极调节都很重要。我们已经成功地在出芽酵母中研究了该系统，该系统使用被称为“RAM网络”的保守的hippo-trc途径来控制细胞分裂和极化生长。在有丝分裂退出机制的密切调控下，该途径直接驱动转录因子的不对称定位和活性，从而开启参与细胞质分裂最后一步的基因表达。除了决定原始细胞的命运外，出芽酵母的海马-trc信号还促进细胞极性的维持，并调节细胞在快速生长过程中物理扩张所需的蛋白质的翻译。本项目旨在明确Cbk1的调控机制和下游靶点。通过计算和实验相结合的工作，我们发现一种新的“对接基序”肽通过与激酶催化结构域的相互作用将这种Ndr/LATS激酶招募到体内底物。我们已经结晶了Mob2-Cbk1复合物并解决了它的结构，这是第一个针对Ndr/LATS激酶的，并将利用这些信息来指导激酶激活机制的分析。我们将定义对接基序如何与Cbk1的激酶结构域结合，分析体内这种相互作用中断所引起的影响，并确定我们在出芽酵母中发现的新型底物对接行为是否也发生在后生动物同源物中。当结合现有的相互作用和磷酸化蛋白质组学数据时，我们对底物对接和共识基序保护的分析至少是高置信度Cbk1靶点数量的三倍。除了进一步分析该通路对mRNA翻译的调控外，我们还将探索这一扩展的调控网络，以更全面地了解这一hippop -trc通路对细胞分裂和形态发生的控制机制。

DESCRIPTION (provided by applicant): Signaling pathways in which GCK group "Mst/hippo" kinases regulate AGC group "Ndr/LATS" kinases are ancient controllers of growth, proliferation, and architecture of eukaryotic cells. Our broad goal is to define the diverse intracellular processes these pathways regulate and determine the mechanisms underlying this control. Forms of Mst/hippo signaling ("hippo-warts" pathways) that suppress metazoan cell proliferation in metazoans by inhibiting YAP/yorkie-related transcriptional co-activators have been the subject of intensive recent interest. However, the distinct and highly conserved "hippo-trc" form of this pathway in which large "furry" related proteins mediate Mst/hippo kinase activation of Ndr/tricornered kinases have dramatically different functions; comparatively little is known about them, and they are the focus of this project. The hippo-trc pathways are important for polarized growth and organization of cellular extensions, neuron morphogenesis, mitotic spindle organization, and positive regulation of cell proliferation. We have successfully studied the system in budding yeast, which use a conserved hippo-trc pathway known as the "RAM network" to control cell division and polarized growth. Under close regulation by mitotic exit machinery, this pathway directly drives asymmetric localization and activity of a transcription factor that turns on expression of genes involved in the final step of cytokinesis. In addition to this primordial cell fate decision, budding yeast hippo-trc signaling promotes maintenance of cell polarity and regulates translation of proteins required for physical expansion of the cell during rapid growth. This project aims to define the regulatory mechanisms and downstream targets of Cbk1. Through combined computational and experimental work we have discovered that a novel "docking motif" peptide recruits this Ndr/LATS kinase to in vivo substrates through interaction with the kinase catalytic domain. We have crystallized the Mob2-Cbk1 complex and solved its structure, the first for any Ndr/LATS kinase, and will use this information to guide analysis of the kinase's activation mechanisms. We will define how the docking motif binds to Cbk1's kinase domain, analyze effects caused by disruption of this interaction in vivo, and determine if the novel substrate docking behavior we have discovered in budding yeast also occurs with metazoan orthologs. When combined with existing interaction and phosphoproteomic data, our analysis of substrate docking and consensus motif conservation at least triples the number of high confidence Cbk1 targets. In addition to extending our analysis of the pathway's regulation of mRNA translation, we will explore this expanded regulatory network to gain a more comprehensive mechanistic understanding of this hippo-trc pathway's control of cell division and morphogenesis.