Characterizing novel functions of conserved NIMA family kinases

 描述（由申请人提供） 尽管NIMA相关激酶（NEK）家族的成员已广泛涉及人类疾病，包括癌症以及肾和心血管缺陷，但它们的生理底物在很大程度上是未知的。此外，对NEK信号通路的上游和下游成分或调节NEK活性的蛋白质知之甚少。这导致了我们对这个生物医学相关和高度保守的蛋白激酶家族的分子和细胞功能的认识存在根本性的空白。长期的目标是表征最近发现的和潜在的广泛的功能NEKs在细胞内运输和细胞外基质重塑。本实验的目的是研究NEKL-2/NEK 8和NEKL-3/NEK 6/7在C. elegans发育和人类细胞培养系统。核心假设是NEK通过与细胞骨架的组分（包括微管（MT）相关蛋白、分子马达和作用网络的调节剂）相互作用来调节内吞作用和囊泡运输。强有力的初步数据支持两个具体目标：1）阐明NEKL-2和NEKL-3控制贩运的机制; 2）鉴定NEKL信号通路的分子靶标和组分。在目标1下，将表征NEKL在细胞内运输和细胞骨架区室中的定位，并且将确定NEKL在调节囊泡运输和细胞骨架组织和动力学中的特定细胞功能。此外，锚蛋白重复序列（AR）结合伙伴的NEKL作为信号支架的假设将进行测试。最后，将进行一组测定以测试NEK 6/7、NEK 8和相关AR蛋白在哺乳动物细胞中的运输功能中的作用。在目标2下，将进行经过验证的RNAi喂养筛选，以确定NEKL信号网络的组件和候选目标。此外，最近开发的强大的化学遗传蛋白质组学策略，这是很好的支持，初步的数据，将被用来直接确定在体内的NEKL基板。将对选定的经验证的 目标，以确定他们的功能，贩运和细胞骨架组织下游的NEK。该方法是创新性的，因为其他研究组尚未应用强大的正向遗传学方法来研究NEK激酶，并且因为NEK在细胞内运输中的功能完全未知。此外，所提出的化学遗传学策略来鉴定底物以前还没有应用于C. elegans或任何系统中NEK的研究。所提出的研究是重要的，因为人NEK广泛涉及人类疾病，并已被建议作为药物靶点，但它们在间期的作用还不清楚，在大多数情况下，它们的生理底物也不清楚。作为一个额外的好处，这些研究将导致以前未知的，但保守的细胞内运输和细胞外基质重塑，这两者都是高度相关的人类发育，健康和疾病的监管机构的特点。

 DESCRIPTION (provided by applicant) Although members of the NIMA-related kinase (NEK) family have been implicated extensively in human dis- eases including cancer and renal and cardiovascular defects, their physiological substrates are largely un- known. Moreover, little is known about the upstream and downstream components of NEK signaling pathways or about the proteins that modulate NEK activities. This has led to a fundamental gap in our knowledge concerning the molecular and cellular functions of this biomedically relevant and highly conserved family of protein kinases. The long-term goal is to characterize a recently discovered and potentially widespread function for NEKs in intracellular trafficking and extracellular matrix remodeling. The objective of this application isto characterize the trafficking functions of NEKL-2/NEK8 and NEKL-3/NEK6/7 during C. elegans development and in human cell culture systems. The central hypothesis is that NEKs regulate endocytosis and vesicle trafficking through interactions with components of the cytoskeleton including microtubule (MT)-associated proteins, molecular motors, and regulators of the acting network. Strong preliminary data support the two specific aims: 1) Elucidate the mechanisms by which NEKL-2 and NEKL-3 control trafficking; and 2) Identify molecular targets and components of the NEKL signaling pathway. Under Aim 1, the localization of NEKLs to intracellular traf- ficking and cytoskeletal compartments will be characterized, and the specific cellular functions of NEKLs in regulating vesicular trafficking and cytoskeletal organization and dynamics will be determined. In addition, the hypothesis that ankyrin repeat (AR) binding partners of NEKLs act as signaling scaffolds will be tested. Finally, a panel of assays will be carried out to test the rles of NEK6/7, NEK8, and associated AR proteins in trafficking functions in mammalian cells. Under Aim 2, proven RNAi-feeding screens will be carried out to identify components and candidate targets of the NEKL signaling networks. In addition, a recently developed powerful chemical genetic-proteomic strategy, which is well supported by preliminary data, will be used to directly identify in vivo substrates of the NEKLs. Follow-up studies will be carried out on select validated targets to deter- mine their functions in trafficking and cytoskeletal organization downstream of the NEKs. The approach is in- novative because other groups have not applied powerful forward genetic methods to study NEK kinases and because the function of NEKs in intracellular trafficking is entirely unknown. In addition the proposed chemical- genetic strategy to identify substrates has not been previously applied to C. elegans or to the study of NEKs in any system. The proposed research is significant because human NEKs are widely implicated in human dis- eases and have been suggested as drug targets, but their roles during interphase are not well understood nor, in most cases, are their physiological substrates known. As an additional benefit, these studies will lead to the characterization of previously unknown but conserved regulators of intracellular trafficking and extracellular matrix remodeling, both of which are highly relevant to human development, health, and disease.