Kinesin-2 Regulation of GLI Function in Hedgehog Signal Transduction

 描述(由申请人提供)：Hedgehog(HH)信号通路对胚胎发育过程中的组织模式和器官形成以及成年组织的动态平衡、更新和再生至关重要。相反，HH途径功能异常会导致许多发育疾病和出生缺陷，并导致越来越多的癌症。Gli蛋白(GLI1-3)是HH途径下游必需的转录效应物，具有转录激活和抑制功能。最近的研究表明，初级纤毛是HH途径活性的重要调节者，部分是通过纤毛靶标和GLI蛋白的运输。然而，在我们理解这些蛋白质通过纤毛运输的机制以及这种运输对HH途径激活下游的GLI蛋白功能的影响方面，存在着关键的差距。在这里，我们建议回答以下问题：是什么调节了GLI蛋白的纤毛靶向和运输，以及这如何影响GLI的转录活性？本研究的长期目标是了解在发育和疾病的HH信号转导过程中，HH通路组件的纤毛运输如何影响其功能。这项建议的目的是准确地确定GLI蛋白通过纤毛运输的机制，以及这种运输对GLI介导的HH信号的功能影响。我们的数据表明，GLI蛋白选择性地与运动蛋白Kinesin-2家族的成员相互作用，破坏这些相互作用显著影响GLI蛋白的功能。因此，我们假设KAP3以及KIF3A、KIF3B和KIF17是GLI蛋白纤毛运输所必需的，并且这种运输是GLI加工和功能所必需的。这一建议的基本原理是，对GLI蛋白纤毛运输的更深层次的了解将显著促进针对越来越多的纤毛疾病和HH驱动的病理的新疗法的开发，在这些疾病中，去调节HH信号促进疾病的发生和发展。为了检验我们的假设，我们提出了两个具体目标。在目标1中，我们将：1)确定GLI蛋白与异三聚体Kinesin-2马达复合体的物理相互作用，2)确定异三聚体Kinesin-2马达在调节GLI蛋白亚细胞定位和加工中的作用，以及3)研究干扰KIF3-KAP3-GLI相互作用对HH信号转导的功能后果。在目标2中，我们将：1)定义GLI蛋白和同源二聚体激动素-2马达KIF17之间的新的相互作用；2)阐明干扰GLI-KIF17相互作用对GLI蛋白定位、加工和活性的影响；3)研究KIF17在体内GLI介导的小脑祖细胞增殖中的新作用。这项拟议的研究将利用体外(生化、成像和细胞信号分析)和体内(小鼠遗传学和鸡卵电穿孔)相结合的方法来确定Kinesin-2与GLI蛋白的相互作用如何影响HH途径功能。这项工作将定义调节HH途径功能的新机制，并将显著影响我们对HH驱动的发育疾病和癌症的理解。

 DESCRIPTION (provided by applicant): The Hedgehog (HH) signaling pathway is vital for tissue patterning and organ formation during embryogenesis, as well as adult tissue homeostasis, renewal and regeneration. In contrast, aberrant HH pathway function results in numerous developmental diseases and birth defects, and is responsible for a growing number of cancers. GLI proteins (GLI1-3) are essential downstream transcriptional effectors of the HH pathway, with both transcriptional activator and repressor functions. Recent work indicates that primary cilia are essential regulators of HH pathway activity, in part through the ciliary targetin and trafficking of GLI proteins. There are critical gaps, however, in our understanding of the mechanisms by which these proteins traffic through cilia, and the consequences that this trafficking has on GLI protein function downstream of HH pathway activation. Here we propose to answer the following questions: what regulates the ciliary targeting and trafficking of GLI proteins, and how does this impact GLI transcriptional activity? The long-term goal of this research is to understand how ciliary transport of HH pathway components affects their function during HH signal transduction in development and disease. The objective of this proposal is to precisely define the mechanisms by which the GLI proteins traffic through cilia and the functional consequences of this transport on GLI-mediated HH signaling. Our data indicate that GLI proteins selectively interact with members of the kinesin-2 family of motor proteins, and that disrupting these interactions significantly impacts GLI protein function. Thus, we hypothesize that the kinesin-associated protein, KAP3, as well as the kinesin-2 motors, KIF3A, KIF3B and KIF17, are essential for ciliary trafficking of GLI proteins, and that this transport is necessary or proper GLI processing and function. The rationale for this proposal is that a deeper mechanistic understanding of the ciliary trafficking of GLI proteins will significantly inform the development f novel therapies for a growing number of ciliopathies and HH-driven pathologies where de-regulated HH signaling promotes disease initiation and progression. To test our hypothesis, we propose two specific aims. In Aim 1 we will: 1) define the physical interactions of GLI proteins with the heterotrimeric kinesin-2 motor complex, 2) determine the role of heterotrimeric kinesin-2 motors in regulating the subcellular localization and processing of GLI proteins, and 3) investigate the functional consequences of disrupting KIF3-KAP3-GLI interactions on HH signal transduction. In Aim 2 we will: 1) define novel interactions between GLI proteins and the homodimeric kinesin-2 motor, KIF17, 2) elucidate the consequences of disrupting GLI-KIF17 interactions on GLI protein localization, processing and activity, and 3) investigate a novel role for KIF17 in GLI-mediated cerebellar progenitor proliferation in vivo. The proposed research will utilize a combination of in vitro (biochemical, imaging, and cell signaling assays) and in vivo (mouse genetics and chicken in ovo electroporations) approaches to define how kinesin-2 interactions with GLI proteins affects HH pathway function. This work will define new mechanisms that regulate HH pathway function, and will significantly impact our understanding of HH-driven developmental diseases and cancers.