Protein kinase A/Hedgehog pathway interaction at the primary cilium

初级纤毛的蛋白激酶 A/Hedgehog 通路相互作用

• 批准号: 8241140
• 负责人: JAMES A WASCHEK
• 金额: $ 7.7万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8241140
• 关键词: A kinase anchoring protein; Adenylate Cyclase; Agonist; Biological Assay; Brain; Cell surface; Cells; Cerebellar Neoplasms; Cerebellum; Chimeric Proteins; Cilia; Consensus; Cultured Cells; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; Cytosol; Development; Dominant-Negative Mutation; Environment; Erinaceidae; Event; Experimental Models; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Future; G-Protein-Coupled Receptors; Goals; Growth; Health; Heterotrimeric GTP-Binding Proteins; Human; Knowledge; Letters; Ligands; Malignant Neoplasms; Mammals; Measures; Molecular; Movement; Mutate; NIH 3T3 Cells; Neoplasm Metastasis; Organ; Organelles; Pathway interactions; Peptides; Phosphorylation; Phosphorylation Site; Pituitary Gland; Play; Post-Translational Protein Processing; Process; Protein Engineering; Protein Kinase A Inhibitor; Proteins; Reagent; Regulation; Research; Role; Signal Transduction; Signaling Protein; Site; Testing; Therapeutic Agents; Transducers; Universities; Vertebrates; cancer type; cell growth regulation; clinically relevant; design; genetic manipulation; improved; knock-down; medulloblastoma; novel; overexpression; smoothened signaling pathway; trafficking; transcription factor; treatment effect

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.

描述（由申请人提供）：Hedgehog （Hh）通路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。研究表明，Hh信号严重依赖于初级纤毛，这是一种从细胞表面延伸到周围环境的天线状细胞器。然而，在分子水平上，该途径的不同组分如何在纤毛上相互作用，人们知之甚少。本应用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）负调控Hh通路中的作用。主要假设是Hh通路受到两个独立的、差异调节的PKA池的负调节，一个隔离在纤毛中，负责在Hh配体缺失时保持Hh通路沉默，另一个在细胞质中，由异源三聚体G蛋白偶联受体调节。这一假设将通过基因操作将各种蛋白质靶向培养细胞的纤毛或纤毛外区室，通过共聚焦荧光显微镜观察它们的亚细胞区室化，然后分析它们对PKA活性和Hh调节的转录因子Gli3的翻译后修饰的影响，Gli3是一种富含纤毛的蛋白质，PKA对其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传导调节的理解。更具体地说，这些结果有望为纤毛中功能齐全的磷酸化途径提供证据，鉴于纤毛细胞器内存在极端的空间限制，这是值得注意的。