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不同的细胞内过程，这些途径调节和确定这种控制的基础机制。MST/河马信号通路通过抑制YAP/York kie相关的转录共激活因子来抑制后生动物后生动物细胞的增殖，是最近引起人们强烈兴趣的主题。然而，该途径的独特和高度保守的“河马-TRC”形式，其中大的“毛茸茸的”相关蛋白介导的MST/河马蛋白激活的NDR/三角蛋白激酶有截然不同的功能；相对较少的人知道它们，他们是本项目的重点。HIPPO-TRC通路对细胞延伸的极化生长和组织、神经元形态发生、有丝分裂纺锤体组织和细胞增殖的正向调节具有重要作用。我们已经成功地在萌芽酵母中研究了这个系统，它使用一条保守的河马-TRC途径，被称为“RAM网络”，来控制细胞分裂和极化生长。在有丝分裂退出机制的严密调控下，该途径直接驱动转录因子的不对称定位和活性，从而启动参与胞质分裂最后一步的基因的表达。除了这种原始的细胞命运决定之外，发芽酵母河马-TRC信号促进了细胞极性的维持，并调节了快速生长期间细胞物理扩张所需蛋白质的翻译。该项目旨在确定Cbk1的调控机制和下游目标。通过计算和实验相结合的工作，我们发现一种新型的“对接基序”多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上。我们已经结晶了Mob2-Cbk1复合体，并解决了它的结构，这在任何NDR/LATS激酶中都是第一次，并将使用这些信息来指导对该激酶激活机制的分析。我们将定义对接基序如何与CBK1的S激酶结构域结合，分析在体内这种相互作用中断所造成的影响，并确定我们在芽殖酵母中发现的新的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蛋白质组数据时，我们对底物对接和共识基序保守的分析至少使高置信度Cbk1靶标的数量增加了两倍。除了扩展我们对该途径对mRNA翻译调控的分析外，我们还将探索这一扩大的调控网络，以更全面地从机制上了解这一河马-TRC途径对细胞分裂和形态发生的控制。

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.