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。我们的广泛目标是确定这些途径调节的不同细胞内过程，并确定这种控制的机制。通过抑制雅普/约基犬相关的转录共激活因子来抑制后生动物中后生动物细胞增殖的Mst/hippo信号传导形式（“疣”途径）已经成为最近强烈兴趣的主题。然而，这种途径的独特和高度保守的“E-trc”形式，其中大的“毛茸茸的”相关蛋白介导的Mst/河马激酶激活的Ndr/tricorned激酶具有显着不同的功能，相对较少的是知道他们，他们是这个项目的重点。该途径是重要的极化生长和组织的细胞延伸，神经元形态发生，有丝分裂纺锤体的组织，和积极调节细胞增殖。我们已经成功地在芽殖酵母中研究了该系统，芽殖酵母使用被称为“RAM网络”的保守的p53-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，该转录因子开启参与胞质分裂最后步骤的基因的表达。除了这种原始细胞的命运决定，芽殖酵母的p53-trc信号促进细胞极性的维持，并调节蛋白质的翻译所需的物理膨胀的细胞在快速生长。该项目旨在确定Cbk 1的调控机制和下游靶点。通过结合计算和实验工作，我们已经发现，一种新的“对接基序”肽招聘这Ndr/LATS激酶在体内底物通过与激酶催化结构域的相互作用。我们已经使Mob 2-Cbk 1复合物结晶并解决了其结构，这是第一个针对任何Ndr/LATS激酶的结构，并将使用这些信息来指导激酶激活机制的分析。我们将定义如何对接基序结合到Cbk 1的激酶结构域，分析这种相互作用在体内破坏所造成的影响，并确定我们在芽殖酵母中发现的新底物对接行为是否也发生在后生动物的直系同源物中。当结合现有的相互作用和磷酸化蛋白质组学数据，我们的分析底物对接和共识基序的保守性至少三倍的高置信度Cbk 1目标的数量。除了扩展我们对mRNA翻译途径调控的分析外，我们还将探索这种扩展的调控网络，以更全面地了解这种p53-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.