Signaling Mechanisms and Cellular Functions of a Ciliopathy-Associated Protein Kinase

纤毛病相关蛋白激酶的信号传导机制和细胞功能

• 批准号: 10210778
• 负责人: Zheng Fu
• 金额: $ 33.27万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10210778
• 关键词: Address; Adenylate Cyclase; Affect; Animal Model; Apical; Binding; C-terminal; Cell model; Cell physiology; Cell surface; Cells; Chemicals; Cilia; Cilium Microtubule; Clinical; Cues; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA Sequence Alteration; Data; Defect; Development; Disease; Enzymes; Epilepsy; Esthesia; Event; Exhibits; Functional disorder; Genes; Genetic Diseases; Genetic Engineering; Growth Factor; Homeostasis; Human; Human Genetics; Impairment; Knowledge; Length; Link; MAP Kinase Gene; Mammalian Cell; Mediating; Microtubules; Molecular; Motor; Mus; Mutation; Organelles; Outcome; PP5 protein-serine-threonine phosphatase; Pathogenicity; Perinatal mortality demographics; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Point Mutation; Production; Protein Dephosphorylation; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Regulation; Reporting; Research; Research Proposals; Role; Scaffolding Protein; Sensory; Signal Pathway; Signal Transduction; Site; Structure; Surface; Testing; Tissues; Variant; Yin-Yang; base; body system; cell motility; ciliopathy; cilium biogenesis; human disease; in vivo; innovation; katanin; kinetosome; loss of function; mutant; novel; null mutation; organ growth; response; smoothened signaling pathway; tissue-factor-pathway inhibitor 2

Project Summary Ciliopathies comprise an expanding group of human disorders associated with genetic mutations causing cilia dysfunction. Cilia can be divided into motile and non-motile (primary) forms. The primary cilium is a microtubule (MT)-based organelle that protrudes from the apical surface of nearly every mammalian cell and plays a critical role in chemical sensation, signal transduction, and control of various cellular functions. To date, there are still major gaps in our knowledge about the dynamic structure and function of the primary cilium, and the underlying molecular basis of ciliopathies. Our research proposal is focused on elucidating the molecular mechanism by which pathogenic mutations in the human CILK1 (ciliogenesis associated kinase 1) gene cause ciliopathies. CILK1 encodes a serine/threonine protein kinase that negatively regulates cilia length and ciliogenesis. Three significant questions about CILK1 remain to be addressed. First, what is the identify of CILK1 substrates that relate to its ciliopathy phenotype? Our data challenged the current view that MT- associated motor KIF3A is the CILK1 substrate responsible for the ciliopathy phenotype by showing that disrupting CILK1 phosphorylation of KIF3A in vivo did not reproduce CILK1 mutant phenotype. In this continuation project, we hypothesize that CILK1 suppresses ciliogenesis by phosphorylating a novel MT- associated ciliary protein and promoting MT disassembly. Second, how is CILK1 activity regulated in the primary cilium? CILK1 requires phosphorylation of its MAPK-like TDY motif for full activation, but growth factors have little stimulatory effect on its activity. Our new preliminary data shows that reducing intracellular cAMP stimulates CILK1. We hypothesize that CILK1 activity is negatively regulated by ciliary cAMP-dependent phosphorylation. Third, how CILK1 human disease variants impact cilia function and signaling and tissue development? We observed human disease variants in the non-catalytic C-terminal domain (CTD) of CILK1 that retain CILK1 catalytic activity but produce a loss-of-function effect on suppression of ciliogenesis. We hypothesize that CILK1 variants in the CTD perturb CILK1 localization and substrate recognition, thereby compromising its ability to suppress ciliogenesis. We propose three specific aims to test these hypotheses. Aim 1 will determine how CILK1 signals through a novel signaling pathway to control cilia length and ciliogenesis. Aim 2 will determine how cAMP inhibits CILK1 to elongate cilia and promote ciliogenesis. Aim 3 will determine the impact of CILK1 pathogenic variants on substrate phosphorylation, cilia function, Hedgehog signaling, and tissue development. The significance of this project derives from human ciliopathies that have an expanding disease spectrum with devastating clinical outcomes. Our studies are innovative in using novel genetically engineered animal and cell models to elucidate new mechanisms that control cilia function and signaling. Our research will exert strong impact on basic knowledge about the primary cilium and significantly advance our understanding of the disease mechanisms underlying human ciliopathies.

项目摘要 纤毛病包括与遗传突变相关的一组扩展的人类疾病，导致纤毛 功能障碍。纤毛可以分为机动和非运动形式。主要纤毛是微管 （MT）的细胞器几乎从每个哺乳动物细胞的顶部表面突出并发挥着关键 在化学感觉，信号转导和控制各种细胞功能中的作用。迄今为止，仍然有 我们对主要纤毛的动态结构和功能的知识的主要差距， 纤毛病的基本分子基础。我们的研究建议的重点是阐明分子 人CILK1中的致病突变（纤毛发生相关激酶1）基因引起的机制 纤毛病。 CILK1编码丝氨酸/苏氨酸蛋白激酶，该激酶对纤毛长度和 纤毛发生。关于CILK1的三个重要问题仍有待解决。首先，什么是识别 与其纤毛性表型相关的CILK1底物？我们的数据挑战了当前的观点 相关的电动机KIF3A是CILK1底物，负责纤毛病表型 破坏体内KIF3A的CILK1磷酸化不会再现CILK1突变体表型。在这个 延续项目，我们假设CILK1通过磷酸化新型MT-抑制了纤毛发生 相关的睫状蛋白并促进MT拆卸。其次，如何调节CILK1活性 原发性纤毛？ CILK1需要其MAPK样TDY基序的磷酸化以进行完全激活，但生长因子 对其活性几乎没有刺激作用。我们的新初步数据表明，减少细胞内营地 刺激CILK1。我们假设CILK1活性受睫状营依赖性的负调节 磷酸化。第三，CILK1人类疾病变体如何影响纤毛功能，信号传导和组织 发展？我们观察到CILK1的非催化C末端结构域（CTD）中的人类疾病变异 保留CILK1催化活性，但会对抑制纤毛生成产生功能丧失作用。我们 假设CTD扰动CILK1定位和底物识别中的CILK1变体，从而 损害其抑制纤毛发生的能力。我们提出了三个特定的目标来检验这些假设。 AIM 1将确定CILK1如何通过新的信号传导途径来控制纤毛长度和 纤毛发生。 AIM 2将确定CAMP如何抑制CILK1延长纤毛并促进纤毛发生。目标3 将确定CILK1致病变异对底物磷酸化，纤毛功能，刺猬的影响 信号传导和组织发育。该项目的意义来自于人类的纤毛病 疾病范围不断扩大，具有破坏性的临床结果。我们的研究在使用新颖的方面具有创新 基因工程的动物和细胞模型，以阐明控制纤毛功能和 信号。我们的研究将对基本知识产生强大的影响，并显着 促进我们对人纤毛病的疾病机制的理解。