Mechanisms of signal integration in developmental control of organ size and tissue patterning

项目摘要 我们的工作旨在提供新的见解，了解信号机制负责 器官生长的发育控制。我们的初步数据表明，Capicua（Cic）是增长的目标- 控制信号通路，并表明Cic整合了几个上游信号来控制器官大小。 Cic是一种转录抑制蛋白，受细胞外受体酪氨酸激酶（RTK）调节。 信号调节激酶（ERK）通路是参与器官大小控制和组织分化的关键系统之一， 在人类和模型实验动物中的模式化。我们的初步研究发现了一种激酶Minibrain (Mnb)作为一种新的Cic调节剂，其与RTK/ERK信号传导平行作用。ERK信号转导元件，Mnb （人DYRK 1A）和Cic是高度保守的蛋白质，并且它们在人体中水平的改变导致 几种疾病，如Rasopathies，神经退行性疾病和癌症。DYRK 1A已经积极地 研究作为唐氏综合征的致病因素之一，并认为是重要的适当 中枢神经系统的发育和生长。我们在这个应用程序中所追求的一个令人兴奋的假设 是来自ERK和Mnb的信号汇聚在Cic上以调节其作为生长抑制剂的活性。鉴于 人类病理学可由ERK和DYRK 1A信号传导的定量变化引起，因此应用 定量方法，以了解潜在的机制。果蝇提供了一种独特的 有机会在体内进行这种研究。我们建议利用这个强大的实验平台， 对控制生长的发育信号进行了定量和系统的分析。我们将使用- 最先进的蛋白质组学方法，如亲和纯化质谱法（AP-MS），以鉴定结合 Cic在胚胎中的合作伙伴，并分析上游信号如何改变这些相互作用，这反过来又导致 Cic活性的变化。然后，我们将应用定量成像分析系统地测试所识别的 相互作用的合作伙伴参与三个分子结果：Cic降解，核输出， 抑制转录阻遏物活性。此外，我们将研究Cic如何整合来自 ERK和Mnb激酶，通过鉴定和功能验证这些激酶靶向的磷酸化位点， 两种激酶，并通过测试这些激酶对Cic调节和生长控制的相对贡献， 独立的体内测定。总之，我们的多层次实验计划旨在提供以下见解 Cic对RTK/ERK信号解释的分子机制，以及确定这些信号是如何被激活的。 机制与Mnb的输入相交，Mnb是一种新型的Cic调节器。从长远来看，这项工作将推动我们的 了解参与器官大小控制的途径之间的复杂调控关系， 可能为开发ERK、Cic和DYRK 1A相关疗法提供新的靶点。

PROJECT SUMMARY Our work is designed to provide new insights into understanding of signaling mechanisms responsible for the developmental control of organ growth. Our preliminary data have implicated Capicua (Cic) as a target of growth- controlling signaling pathways and suggest that Cic integrates several upstream signals to control organ size. Cic is a transcriptional repressor protein that is regulated by the receptor tyrosine kinase (RTK)-extracellular signal regulated kinase (ERK) pathway, which is one of the key systems involved in organ size control and tissue patterning in humans and model experimental animals. Our preliminary studies have identified a kinase Minibrain (Mnb) as a novel Cic regulator which acts in parallel to RTK/ERK signaling. ERK signaling components, Mnb (human DYRK1A), and Cic are highly conserved proteins, and alterations in their levels in humans result in several diseases, such as Rasopathies, neurodegenerative disorders, and cancer. DYRK1A has been actively investigated as one of the causative factors in Down syndrome, and is thought to be important for proper development and growth of the central nervous system. An exciting hypothesis that we pursue in this application is that signals from ERK and Mnb converge on Cic to regulate its activity as a growth suppressor. Given that human pathologies can result from quantitative changes in ERK and DYRK1A signaling, it is essential to apply quantitative approaches in order to understand the underlying mechanisms. Drosophila offers a unique opportunity to carry out such research in vivo. We propose to use this powerful experimental platform to carry out a quantitative and systems-level analysis of developmental signals controlling growth. We will use state-of- the-art proteomic methods, such as affinity purification mass spectrometry (AP-MS), to identify the binding partners of Cic in the embryo and analyze how upstream signals alter these interactions, which in turn leads to changes in Cic activity. We will then apply quantitative imaging assays to systematically test the identified interacting partners for their involvement in three molecular outcomes: Cic degradation, nuclear export, and inhibition of transcriptional repressor activity. Furthermore, we will investigate how Cic integrates signals from the ERK and Mnb kinases by identifying and functionally validating the phosphorylation sites targeted by these two kinases, and by testing the relative contributions of these kinases to Cic regulation and growth control in four independent in vivo assays. In summary, our multi-level experimental plan is designed to provide insights into the molecular mechanisms of RTK/ERK signal interpretation by Cic, as well as to determine how these mechanisms intersect with the input from Mnb, a novel Cic regulator. In the long term, this work will advance our understanding of the complex regulatory relationships between the pathways involved in organ size control, and may suggest new targets for developing ERK, Cic, and DYRK1A-related therapies.