Steroid-Dependent Changes in the Yorkie Interactome

描述（由申请人提供）：Hippo（或alvador/Warts/Hippo，SWH）通路是一种高度保守的后生动物信号级联反应，可限制果蝇（Drosophila melanogaster）的器官大小，并限制哺乳动物的肿瘤发生。该途径的主要靶标是Yorkie（Yki）/YAP 1转录共激活因子。许多实验室在昆虫细胞、人类细胞和小鼠细胞中研究Hippo及其靶点Yki/Yap 1的工作都围绕着这样一个假设，即该途径的主要调节因子是介导细胞之间相互作用的跨膜分子。根据该模型，Yki/Yap 1的主要作用是充当接触抑制机制的关键靶标，通过该接触抑制机制，膜相关细胞粘附分子“感知”细胞拥挤并抑制Yki/Yap 1以使细胞保持在有丝分裂后状态。 在我们 这个模型是不完整的。由于大多数上皮细胞在其整个生命中与相邻细胞紧密贴壁，细胞：细胞粘附的大波动似乎是通常依赖于Yki/Yap 1的发育生长的不太可能的驱动因素。在未发表的工作中，我们发现了一种新形式的粘附独立Yki调节的生化和遗传证据，在果蝇组织的生理生长中起着重要作用。我们发现，蛋白质介导的细胞反应蜕皮激素，在苍蝇中的主要类固醇激素，也需要细胞：细胞粘附独立的调制YKI活动在正常的生理生长。此外，我们可以将这种Yki调节作用细化为Yki本身与EC应答蛋白Taiman之间的分子相互作用，Taiman的人类同源物类固醇受体辅激活因子-3/乳腺癌扩增因子-1（SRC 3/AIB-1）在多种癌症中扩增。我们对Yorkie-Taiman复合物的初步蛋白质组学分析已经确定了额外的组分，这些组分似乎定义了我们称为Yorkie核相互作用组（YNI）的大分子相互作用网络。我们的长期目标是了解单个YNI蛋白如何影响果蝇中Yki驱动的基因表达，以确定触发YNI内相互作用重排的机制。目前研究的目的是使用尖端的蛋白质组学技术来分析YNI在整个生物体中的大分子组成，并在Ec的存在下，并将其与单个YNI组分在Yki核活性中的作用的遗传分析相结合。 在该建议的第一个目的中，我们将使用定量亲和纯化-质谱（AP-MS）技术来进行在生理环境f椎间盘细胞中的YNI蛋白的更大套件的体内鉴定，另外重点在于鉴定特异性地募集到YNI或在暴露于升高水平的循环Ec之后从其置换的蛋白。在第二个目标中，我们将在果蝇中使用多种遗传工具来评估单个YNI蛋白（在AP-MS实验中鉴定）在控制发育组织中的Yki活性中的作用。

DESCRIPTION (provided by applicant): The Hippo (or alvador/Warts/Hippo, SWH) pathway is a well-conserved metazoan signaling cascade that restricts organ size in the fruit fly Drosophila melanogaster and limits tumorigenesis in mammals. The main target of this pathway is the Yorkie (Yki)/YAP1 transcriptional co-activator. Work from many labs studying Hippo and its target Yki/Yap1 in insect cells, human cells and mouse cells has coalesced around the hypothesis that the main regulators of this pathway are transmembrane molecules that mediate interactions between cells. According to this model, the main role of Yki/Yap1 is to serve as a key target of contact- inhibition mechanisms through which membrane-associated cell adhesion molecules 'sense' cell crowding and inhibit Yki/Yap1 to keep cells in a post-mitotic state. In our view this model is incomplete. As most epithelial cells spend their entire lives closely apposed with adjacent cells, large fluctuations in cell:cell adhesion seem to be an unlikely driver of the developmental growth that is normally dependent on Yki/Yap1. In unpublished work, we have found biochemical and genetic evidence of a novel form of adhesion-independent Yki regulation that plays a significant role in the physiologic growth of Drosophila tissues. We find that protein that mediate the cellular response to Ecdysone, the major steroid hormone in flies, are also required for cell:cell adhesion-independent modulation of Yki activity during normal, physiologic growth. Moreover, we can refine this Yki-regulatory effect to a molecular interaction between Yki itself and the Ec-responsive protein Taiman, whose human homolog Steroid Receptor Coactivator-3/Amplified-In-Breast Cancer-1 (SRC3/AIB-1) is amplified in a wide array of cancers. Our initial proteomic analysis of this Yorkie-Taiman complex has identified additional components that appear to define a macromolecular interaction network we have termed the Yorkie Nuclear Interactome (YNI). Our long-term goal is to understand how individual YNI proteins affect Yki-driven gene expression in Drosophila to identify mechanisms that trigger rearrangement of interactions within the YNI. The aim of the current studies is to use cutting-edge proteomic techniques to analyze the macromolecular composition of the YNI in the whole organism and in the presence of Ec, and to couple this with genetic analyses of the role of individual YNI components in Yki nuclear activity in developing imaginal discs. In the first aim o this proposal, we will use quantitative affinity purification-mass spectrometry (AP-MS) technique to carry out in vivo identification of the larger suite of YNI proteins in the physiologic setting f imaginal disc cells, with an additional focus on identification of proteins that are specifically recruited to the YNI or displaced from it following exposure to elevated levels of circulating Ec. In the second aim, we will use the diverse array of genetic tools in Drosophila to assess the role of individual YNI proteins (identified in the AP- MS experiments) in controlling Yki activity in developing tissues.