Dsh/Dvl Phosphorylation and Signaling Outcome

描述（由申请人提供）：卷曲（Fz）受体通过几种效应途径，特别是Wnt/2-连环蛋白（2-cat）和Fz/平面细胞极性（PCP）信号传导，作为Wnt生长因子的信号转导子。Fz/PCP和Wnt-Fz/2-cat信号传导在胞质因子Dishevelled（Dsh）下游发散。通常这两种途径在相同的组织中起作用，并且信号特异性的严格调节对于正常发育和细胞稳态是必不可少的。在放松管制的情况下，选择错误的Fz途径通常会导致疾病（即PKD或癌症）。虽然这两种途径的分子框架已经建立，但对Wnt/Fz-Dsh信号特异性的调节知之甚少。本申请研究区分Wnt-Fz/2-Cat和Fz/PCP信号传导的信号传导特异性的分子方面。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。具体重点是通过磷酸化调节Dsh。Dsh是两种途径共享的细胞质因子，并通过Fz受体募集到膜上。基于初步的数据，我们假设，Dsh是差异磷酸化的途径特异性的方式。细胞培养、体内和生物化学研究的组合将剖析Dsh上的磷酸化事件，其在Wnt-Fz途径之间切换其功能。具体目标是：1。确定Dsh上的特异性磷酸化事件及其在选择信号传导结果中的作用;和2.鉴定和功能性定义酪氨酸激酶在Dsh调节和途径特异性反应中的作用。我们的质谱研究表明，差异磷酸化的DSH有助于通路选择，我们已经确定了几个激酶作用于DSH。将在体内确定磷酸化事件的生理和医学相关性。这将通过产生针对Dsh中相关磷酸化位点的特异性抗血清来补充。这些可能具有诊断潜力。公共卫生相关性：正确的Wnt信号通路的调节和选择是发育和组织功能和稳态的基本特征。Wnt的失调与许多疾病有关，从纤毛病变和血管生成缺陷到癌症（例如，几种途径组分是肿瘤抑制因子或原癌基因）。本申请解决了Wnt/卷曲信号特异性中的关键组分Dishvelled的分子调控;这里获得的信息对几种医学疾病具有重要意义。

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.