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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

基本信息

批准号：
10398240
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
项目类别：
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/10398240
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

项目摘要

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.

项目概要纤毛病包括一组不断扩大的与引起纤毛的基因突变相关的人类疾病功能障碍。纤毛可分为运动型和非运动型（初级）形式。初级纤毛是微管基于 MT（MT）的细胞器，从几乎每个哺乳动物细胞的顶端表面突出，并发挥着关键作用在化学感觉、信号转导和各种细胞功能的控制中发挥作用。迄今为止，仍有我们对初级纤毛的动态结构和功能的认识存在重大差距，纤毛病的潜在分子基础。我们的研究计划的重点是阐明分子人类 CILK1（纤毛发生相关激酶 1）基因致病突变的机制纤毛病。 CILK1 编码丝氨酸/苏氨酸蛋白激酶，可负向调节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，什么是身份与其纤毛病表型相关的 CILK1 底物？我们的数据挑战了当前的观点，即 MT- 相关运动 KIF3A 是负责纤毛病表型的 CILK1 底物，表明在体内破坏 KIF3A 的 CILK1 磷酸化不会重现 CILK1 突变表型。在这个在后续项目中，我们假设 CILK1 通过磷酸化一种新型 MT- 来抑制纤毛发生相关纤毛蛋白并促进 MT 分解。其次，CILK1的活性是如何被调控的？初级纤毛？ CILK1 需要对其 MAPK 样 TDY 基序进行磷酸化才能完全激活，但生长因子对其活性影响不大。我们新的初步数据表明，减少细胞内 cAMP 刺激 CILK1。我们假设 CILK1 活性受到睫状体 cAMP 依赖性的负调节磷酸化。第三，CILK1人类疾病变异如何影响纤毛功能和信号传导以及组织发展？我们观察到 CILK1 非催化 C 端结构域 (CTD) 中的人类疾病变异保留 CILK1 催化活性，但会产生抑制纤毛发生的功能丧失效应。我们假设 CTD 中的 CILK1 变异扰乱了 CILK1 定位和底物识别，从而损害其抑制纤毛发生的能力。我们提出三个具体目标来检验这些假设。目标 1 将确定 CILK1 如何通过新的信号通路发出信号来控制纤毛长度和纤毛发生。目标 2 将确定 cAMP 如何抑制 CILK1 以延长纤毛并促进纤毛发生。目标 3 将确定 CILK1 致病性变异对底物磷酸化、纤毛功能、Hedgehog 的影响信号传导和组织发育。该项目的重要性源于人类纤毛病不断扩大的疾病谱，带来毁灭性的临床结果。我们的研究在使用新颖性方面具有创新性基因工程动物和细胞模型，以阐明控制纤毛功能和发信号。我们的研究将对初级纤毛的基础知识产生重大影响，并显着增进我们对人类纤毛病潜在疾病机制的理解。