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

项目摘要 纤毛病包括一组不断扩大的人类疾病与基因突变引起的纤毛 功能障碍纤毛可分为能动和非能动（初级）形式。初级纤毛是一个微管 （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突出， 在化学感觉、信号传导和控制各种细胞功能中的作用。迄今为止， 我们对初级纤毛的动态结构和功能的认识存在重大差距， 纤毛病变的潜在分子基础我们的研究计划集中在阐明 人CILK1（纤毛发生相关激酶1）基因致病突变导致 纤毛病变CILK1编码丝氨酸/苏氨酸蛋白激酶，其负调节纤毛长度， 纤毛发生关于CILK1的三个重要问题仍有待解决。第一，什么是身份 CILK1底物与其纤毛病变表型有关？我们的数据挑战了目前的观点，即MT- 相关的运动KIF3A是CILK1底物，负责纤毛病变表型， 在体内破坏KIF3A的CILK1磷酸化不能再现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- 相关的纤毛蛋白和促进MT分解。第二，CILK1活性如何调节， 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 对它的活性几乎没有刺激作用。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 磷酸化第三，CILK1人类疾病变体如何影响纤毛功能和信号传导以及组织 发展？我们在CILK1的非催化C-末端结构域（CTD）中观察到人类疾病变体， 其保留了CILK 1催化活性但对纤毛发生抑制产生功能丧失作用。我们 假设CTD中CILK1变体干扰CILK1定位和底物识别，从而 损害其抑制纤毛发生的能力。我们提出了三个具体的目标来测试这些假设。 目的1将确定CILK1如何通过一种新的信号传导途径来控制纤毛长度， 纤毛发生目的2将确定cAMP如何抑制CILK 1延长纤毛和促进纤毛发生。目标3 将确定CILK1致病性变体对底物磷酸化、纤毛功能、Hedgehog 信号传导和组织发育。这个项目的意义来自于人类的纤毛病变， 不断扩大的疾病谱带来毁灭性的临床结果。我们的研究是创新的， 基因工程动物和细胞模型，以阐明控制纤毛功能的新机制， 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， 推进我们对人类纤毛病变的疾病机制的理解。

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.