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

项目摘要 纤毛疾病包括与引起纤毛的基因突变相关的人类疾病的不断扩大的群体 功能障碍。纤毛可分为运动型和非运动型(初级)。初级纤毛为微管。 以(MT)为基础的细胞器，从几乎每一个哺乳动物细胞的顶端表面伸出，并发挥关键的 在化学感觉、信号转导和控制各种细胞功能中的作用。到目前为止，仍然有 我们对初级纤毛的动态结构和功能的认识的主要差距，以及 纤毛疾病的潜在分子基础。我们的研究计划集中在阐明分子 人纤毛发生相关蛋白1基因致病突变的致病机制 纤毛病。CILK1编码丝氨酸/苏氨酸蛋白激酶，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 与其睫毛病变表型相关的CILK1底物？我们的数据挑战了目前的观点，即MT- 相关运动KIF3A是CILK1底物，通过研究表明，CILK1与纤毛病变表型有关 体内干扰KIF3A的CILK1磷酸化不能复制CILK1突变表型。在这 继续项目，我们假设CILK1通过磷酸化一种新的MT-1来抑制纤毛发生。 相关纤毛蛋白和促进MT分解。第二，CILK1活性是如何在 初级纤毛？CILK1需要其MAPK样TDY基序的磷酸化才能完全激活，但生长因子 对其活性的刺激作用不大。我们新的初步数据显示，减少细胞内cAMP 刺激CILK1。我们假设CILK1活性受纤毛cAMP依赖的负性调节 磷酸化。第三，CILK1人类疾病变异如何影响纤毛功能、信号和组织 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 保留了CILK1的催化活性，但在抑制纤毛发生方面产生了功能丧失的效果。我们 假设CTD中的CILK1变体扰乱了CILK1的定位和底物识别，从而 影响其抑制纤毛形成的能力。我们提出了三个具体目标来检验这些假说。 目标1将确定CILK1是如何通过一种新的信号通路来控制纤毛长度和 纤毛发生。目的2将确定cAMP如何抑制CILK1延长纤毛和促进纤毛发生。目标3 将确定CILK1致病变异体对底物磷酸化、纤毛功能、刺猬的影响 信号和组织发育。这个项目的意义来自于人类纤毛疾病 不断扩大的疾病谱具有毁灭性的临床后果。我们的研究在使用小说方面是创新的 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本知识产生强烈的影响，并具有重要意义 促进我们对人类纤毛疾病潜在的疾病机制的理解。

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.