Protein kinase A/Hedgehog pathway interaction at the primary cilium

描述（由申请人提供）：Hedgehog（Hh）途径涉及哺乳动物发育的许多方面，并且它还涉及多种类型癌症的生长和转移。 已经表明，Hh信号传导严重依赖于初级纤毛，初级纤毛是从细胞表面延伸到周围环境中的触角样细胞器。 然而，很少有人知道，在分子水平上，不同的组成部分的途径相互作用的纤毛。 本申请的总体目标是阐明初级纤毛在脊椎动物中通过蛋白激酶A（PKA）负调节Hh途径中的作用。 主要的假设是，Hh途径受到两个独立的，差异调节池PKA，一个隔离在纤毛和负责保持沉默的Hh途径时，Hh配体是不存在的，和其他在细胞质和调节异源三聚体G蛋白偶联受体的负调制。 通过遗传操作将各种蛋白质靶向培养细胞中的睫状体或睫状体外区室，通过共聚焦荧光显微镜观察它们的亚细胞区室化，然后测定它们对PKA活性和对Hh调节的转录因子Gli 3的翻译后修饰的影响，一种富含纤毛的蛋白质，其被PKA磷酸化和蛋白水解裂解被Hh信号阻断。 这些研究有可能大大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，预期结果将提供证据的纤毛，这是显着的极端空间限制，存在于这个细胞器内的一个功能齐全的磷酸化途径。 项目叙述：Hedgehog（Hh）通路参与器官发育的许多方面，并且它还与多种类型的癌症的生长和转移有关。 由Hh通路触发的分子事件被蛋白激酶A（PKA）拮抗，但其意义尚不清楚，阻碍了靶向PKA的有用药理学试剂的开发。 该应用测试了关于Hh/PKA相互作用的新假设，如果该假设被证明是正确的，则有望为开发全新的治疗剂组以阻断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.