Steroid-Dependent Changes in the Yorkie Interactome

描述(申请人提供)：河马(或阿尔瓦多/瘤/河马，SWH)途径是一个保守的后生动物信号级联，限制果蝇黑腹果蝇的器官大小，并限制哺乳动物的肿瘤发生。该途径的主要靶点是约克(Yki)/YAP1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河马及其靶标yki/yap1的工作围绕着这样一个假设：这一途径的主要调节因子是调节细胞间相互作用的跨膜分子。根据这个模型，yki/YAP1的主要作用是作为接触抑制机制的关键靶点，通过接触抑制机制，膜相关细胞黏附分子感知细胞拥挤，抑制yki/YAP1，使细胞保持在有丝分裂后状态。在我们的 查看此模型不完整。由于大多数上皮细胞的一生都与相邻细胞紧密相对，细胞：细胞黏附的大幅波动似乎不太可能是通常依赖于yki/yap1的发育生长的驱动因素。在未发表的工作中，我们发现了一种新形式的黏附非依赖性yki调控的生化和遗传学证据，这种调控在果蝇组织的生理生长中发挥着重要作用。我们发现，介导细胞对蜕皮激素(果蝇中主要的类固醇激素)反应的蛋白质也是细胞所必需的：在正常的生理生长过程中，细胞黏附不依赖于yki活动的调节。此外，我们可以将这种yki调节作用细化为yki本身与EC反应蛋白Taiman之间的分子相互作用，Taiman的人类同源类固醇受体共激活因子-3/放大的乳腺癌-1(SRC3/AIB-1)在多种癌症中被扩增。我们对这个约克-泰曼复合体的初步蛋白质组学分析已经确定了额外的成分，这些成分似乎定义了一个大分子相互作用网络，我们称之为约克核相互作用组(YNI)。我们的长期目标是了解个别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。本研究的目的是利用最先进的蛋白质组学技术分析YNI在整个生物体和EC存在下的大分子组成，并将其与YNI组分在YKI核活动中的作用的遗传分析相结合。在这项建议的第一个目的，我们将使用定量亲和纯化-质谱学(AP-MS)技术，在体内鉴定更大的一套YNI蛋白在生理环境中的想象盘细胞，另外一个重点是鉴定的蛋白质，特别是招募到YNI或取代它暴露在高水平的循环EC。在第二个目标中，我们将使用果蝇中不同的遗传工具来评估单独的YNI蛋白(在AP-MS实验中确定)在控制发育组织中YKI活性方面的作用。

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 In unpublished work, we have found biochemical and genetic evidence of a novel form of adhesion-independent Yki regulation that plays a significant role in the physiologic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.