Kinesin-2 Regulation of GLI Function in Hedgehog Signal Transduction

Kinesin-2 对 Hedgehog 信号转导中 GLI 功能的调节

• 批准号: 9441784
• 负责人: Benjamin Allen
• 金额: $ 31.75万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9441784
• 关键词: Activator Appliances; Adult; Affect; Anger; Biochemical; Biochemistry; Biological Assay; Cells; Cellular biology; Chickens; Cilia; Complex; Congenital Abnormality; Data; Development; Disease; Electroporation; Embryonic Development; Equilibrium; Erinaceidae; Family; Future Generations; GLI Family Protein; GLI gene; Genetic; Genetic Transcription; Goals; Homeostasis; Image; In Vitro; Kinesin; Malignant Neoplasms; Mediating; Modeling; Motor; Mus; Natural regeneration; Organ; Organelles; Pathology; Pathway interactions; Pattern; Proteins; Regulation; Research; Resolution; Role; Signal Transduction; Testing; Therapeutic Uses; Tissues; Transcription Coactivator; Transcription Repressor/Corepressor; Transcriptional Activation; Work; anterograde transport; cellular imaging; ciliopathy; developmental disease; developmental genetics; gene repression; genetic approach; hedgehog signal transduction; in vivo; insight; member; novel; novel therapeutics; progenitor; protein function; protein transport; public health relevance; response; smoothened signaling pathway; trafficking; transcription factor

 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.

 描述（由申请人提供）：Hedgehog（HH）信号通路对于胚胎发生期间的组织模式和器官形成以及成体组织稳态、更新和再生至关重要。相比之下，异常的HH通路功能导致许多发育疾病和出生缺陷，并且是导致越来越多的癌症的原因。GLI蛋白（GLI 1 -3）是HH通路的重要下游转录效应子，具有转录激活子和抑制子功能。最近的研究表明，初级纤毛是HH途径活性的重要调节剂，部分通过纤毛靶向和GLI蛋白的运输。然而，在我们对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蛋白功能的影响的理解中存在关键的差距。在这里，我们建议回答以下问题：是什么调节纤毛靶向和运输的GLI蛋白，这是如何影响GLI转录活性？本研究的长期目标是了解HH通路组分的纤毛转运如何影响其在发育和疾病中HH信号转导过程中的功能。本提案的目的是精确定义GLI蛋白通过纤毛运输的机制以及这种运输对GLI介导的HH信号传导的功能后果。我们的数据表明，GLI蛋白选择性地与驱动蛋白-2家族的马达蛋白的成员相互作用，并且破坏这些相互作用显著影响GLI蛋白的功能。因此，我们假设驱动蛋白相关蛋白KAP 3以及驱动蛋白2马达KIF 3A、KIF 3B和KIF 17对于GLI蛋白的纤毛运输是必需的，并且这种运输是必要的或适当的GLI加工和功能。该提议的基本原理是，对GLI蛋白的纤毛运输的更深入的机制理解将为越来越多的纤毛病和HH驱动的病理学的新疗法的开发提供重要信息，其中去调节的HH信号传导促进疾病的发生和进展。为了验证我们的假设，我们提出了两个具体目标。目标1：1）确定GLI蛋白与异源三聚体驱动蛋白-2马达复合物的物理相互作用，2）确定异源三聚体驱动蛋白-2马达在调节GLI蛋白的亚细胞定位和加工中的作用，和3）研究破坏KIF 3-KAP 3-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）确定GLI蛋白和同型二聚体驱动蛋白-2马达KIF 17之间的新相互作用，2）阐明破坏GLI-KIF 17相互作用对GLI蛋白定位、加工和活性的影响，和3）研究KIF 17在体内GLI介导的小脑祖细胞增殖中的新作用。拟议的研究将利用体外（生物化学，成像和细胞信号传导测定）和体内（小鼠遗传学和鸡卵内电穿孔）方法的组合来定义驱动蛋白2与GLI蛋白的相互作用如何影响HH通路功能。这项工作将定义调节HH通路功能的新机制，并将显著影响我们对HH驱动的发育疾病和癌症的理解。