Protein kinase A/Hedgehog pathway interaction at the primary cilium

描述(申请人提供)：Hedgehog(HH)途径参与哺乳动物发育的许多方面，也与多种类型癌症的生长和转移有关。已经证明，HH信号严重依赖于初级纤毛，初级纤毛是一种类似天线的细胞器，从细胞表面延伸到周围环境。然而，在分子水平上，人们对该途径的不同组成部分如何在纤毛上相互作用知之甚少。这一应用的总体目标是阐明初级纤毛在蛋白激酶A(PKA)对脊椎动物HH途径的负调控中的作用。基本的假设是，HH途径受到两个独立的、不同调节的PKA池的负调控，一个隔离在纤毛中，负责在HH配体缺失时保持HH途径的沉默，另一个在细胞质中，受异源三聚体G蛋白偶联受体的调节。这一假说将通过基因操作将各种蛋白质靶向培养细胞中的纤毛或纤毛外间隔，通过共聚焦荧光显微镜观察它们的亚细胞区划，然后分析它们对PKA活性和HH调节的转录因子Gli3翻译后修饰的影响，Gli3是一种富含纤毛的蛋白质，其被PKA的磷酸化和蛋白水解性切割被HH信号阻断。这些研究有可能极大地提高我们对初级纤毛如何参与细胞信号调节的理解。更具体地说，这些结果有望为纤毛中具有全功能的磷酸化途径提供证据，鉴于该细胞器内存在极端的空间限制，这一点是值得注意的。 项目简介：Hedgehog(HH)途径涉及器官发育的许多方面，也与多种类型癌症的生长和转移有关。蛋白激酶A(PKA)可拮抗HH途径所触发的分子事件，但其意义尚不清楚，阻碍了针对PKA的有用药理药物的开发。这项应用测试了一种关于HH/PKA相互作用的新假说，如果被证明是正确的，有望为开发全新的治疗药物集来阻断HH途径的促癌作用奠定基础。

DESCRIPTION (Provided by Applicant): The Hedgehog (Hh) pathway is involved in many aspects of mammalian development, and it also has been implicated in the growth and metastasis of multiple types of cancer. It has been shown that Hh signaling is critically dependent on the primary cilium, an antenna-like organelle that extends from the cell surface into the surrounding environment. However, little is known about how, on the molecular level, different components of the pathway interact at the cilium. The overall goal of this application is to elucidate the role of primary cilium in the negative regulation of the Hh pathway by protein kinase A (PKA) in vertebrates. The primary hypothesis is that the Hh pathway is subject to negative modulation by two separate, differentially regulated pools of PKA, one sequestered in the cilium and responsible for keeping the Hh pathway silenced when the Hh ligand is absent, and the other in the cytoplasm and regulated by heterotrimeric G protein-coupled receptors. This hypothesis will be tested by targeting various proteins to ciliary or extra-ciliary compartments in cultured cells through genetic manipulation, visualizing their subcellular compartmentalization by confocal fluorescence microscopy, and then assaying their effect on PKA activity and on posttranslational modifications of the Hh regulated transcription factor Gli3, a protein enriched in cilia and whose phosphorylation by PKA and proteolytic cleavage is blocked by Hh signaling. These studies have the potential to greatly improve our understanding of how the primary cilium participates in the regulation of cellular signaling. More specifically, the results are expected to provide evidence for a fully functional phosphorylation pathway in the cilium, which is remarkable in view of extreme spatial constraints that exist within this organelle. PROJECT NARRATIVE: The Hedgehog (Hh) pathway is involved in many aspects of organ development and it also has been implicated in the growth and metastasis of multiple types of cancer. The molecular events triggered by the Hh pathway are antagonized by protein kinase A (PKA), but the significance is poorly understood, hampering the development of useful pharmacologic agents that target PKA. This application tests a novel hypothesis with respect to the Hh/PKA interaction that, if shown to be true, is expected to lay the groundwork for developing entirely new sets of therapeutic agents to block the cancer-promoting actions of the Hh pathway.