Dsh/Dvl Phosphorylation and Signaling Outcome

描述(申请人提供)：卷曲(FZ)受体通过几个效应通路作为WNT生长因子的信号转导，特别是WNT/2-连环蛋白(2-CAT)和FZ/平面细胞极性(PCP)信号。Fz/PCP和Wnt-Fz/2-CAT信号在细胞质因子杂乱(Dsh)下游分化。通常，这两条通路都在同一组织中起作用，对信号特异性的严格调控对于正常发育和细胞动态平衡是必不可少的。在放松管制的情况下，选择错误的FZ途径往往会导致疾病(如PKD或癌症)。虽然这两条通路的分子框架已经建立，但对Wnt/Fz-Dsh信号特异性的调控知之甚少。这项应用研究了区分WNT-FZ/2-Cat和FZ/PCP信号的信号特异性的分子方面。我们将在果蝇(发现这两种途径的模式生物)和哺乳动物细胞培养中解决这一问题。具体的焦点是通过磷酸化来调节Dsh。DSH是一种细胞质因子，由FZ受体募集到细胞膜上，并在两条通路之间共享。根据初步数据，我们假设Dsh以一种途径特异性的方式差异磷酸化。结合细胞培养、体内和生化研究，将剖析Dsh上的磷酸化事件，这些事件将在Wnt-Fz途径之间切换其功能。其具体目的是：1.确定Dsh上的特异性磷酸化事件(S)及其在信号转导结果选择中的作用；2.鉴定并从功能上确定参与Dsh调节和通路特异性反应的酪氨酸激酶。我们的质谱学研究表明，Dsh的差异磷酸化有助于途径的选择，我们已经鉴定了几种作用于Dsh的激酶。磷酸化事件的生理和医学相关性将在体内建立。这将通过产生针对Dsh中相关磷酸化位点的特定抗血清来补充。这些可能具有诊断潜力。公共卫生相关性：调节和选择正确的Wnt信号通路是发育、组织功能和动态平衡的基本特征。Wnt的解除调控与许多疾病有关，从纤毛疾病和血管生成缺陷到癌症(例如，几个通路组件是肿瘤抑制或原癌基因)。这项应用解决了DishvelledWnt/Frizzled一种关键成分的分子调控-信号特异性；这里获得的信息将对几种医学疾病具有很高的意义。

DESCRIPTION (provided by applicant): Frizzled (Fz) receptors act as signal transducers of Wnt growth factors via several effector pathways, notably Wnt/2-catenin (2-cat) and Fz/planar cell polarity (PCP) signaling. Fz/PCP and Wnt-Fz/2-cat signaling diverge downstream of the cytoplasmic factor Dishevelled (Dsh). Often both pathways act in the same tissues, and a tight regulation of signaling specificity is essential for normal development and cellular homeostasis. In deregulated scenarios, selection of the wrong Fz-pathway often leads to disease (i.e. PKD or cancer). Although a molecular framework for both pathways is established, very little is known about the regulation of Wnt/Fz-Dsh signaling specificity. This application investigates the molecular aspects of signaling specificity distinguishing between Wnt-Fz/2-Cat and Fz/PCP signaling. We will address this in Drosophila (the model organism where both pathways were discovered) and in mammalian cell culture. The specific focus is the regulation of Dsh by phosphorylation. Dsh is a cytoplasmic factor shared between both pathways and recruited to the membrane by Fz receptors. Based on preliminary data we hypothesize that Dsh is differentially phosphorylated in a pathway specific manner. A combination of cell culture, in vivo, and biochemical studies will dissect the phosphorylation events on Dsh that switch its function between the Wnt- Fz pathways. The Specific Aims are: 1. To determine specific phosphorylation event(s) on Dsh and their role in the selection of the signaling outcome; and 2. To identify and functionally define Tyrosine kinases acting in Dsh regulation and pathway specific responses. Our mass-spectrometry studies indicated that differential phosphorylation of Dsh contributes to pathway selection and we have identified several kinases acting on Dsh. Physiological and medical relevance of the phosphorylation events will be established in vivo. This will be complemented by the generation of specific antisera to the relevant phosphorylated sites in Dsh. These are likely to have diagnostic potential. PUBLIC HEALTH RELEVANCE: The regulation and selection of the correct Wnt signaling pathways is an essential feature of development and tissue function and homeostasis. Deregulation of Wnt is linked to many diseases, ranging from ciliopathies and angiogenesis defects, to cancer (e.g. several pathway components are tumor suppressors or proto-oncogenes). This application addresses the molecular regulation of Dishvelled a key component in Wnt/Frizzled-signaling specificity; the information acquired here will be of high significance for several medical disorders.