Characterizing novel functions of conserved NIMA family kinases

 描述(由申请人提供) 尽管NIMA相关激酶(NEK)家族的成员广泛参与人类疾病，包括癌症、肾脏和心血管缺陷，但其生理底物很大程度上是未知的。此外，人们对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上的根本差距。长期的目标是描述最近发现的和潜在的广泛的Neks在细胞内运输和细胞外基质重塑中的功能。这项应用的目的是表征NEKL-2/NEK8和NEKL-3/NEK6/7在线虫发育和人类细胞培养系统中的运输功能。中心假说是Neks通过与微管(MT)相关蛋白、分子马达和作用网络调节器等细胞骨架成分的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这两个特定目的：1)阐明NEKL-2和NEKL-3控制贩运的机制；2)确定NEKL信号通路的分子靶点和成分。在目标1下，将确定NEKL对细胞内转运和细胞骨架隔间的定位，并将确定NEKL在调节囊泡运输和细胞骨架组织和动力学方面的特定细胞功能。此外，NEKL的Ankyrin Repeat(AR)结合伙伴作为信号支架的假设将得到验证。最后，将进行一系列检测，以测试NEK6/7、NEK8和相关AR蛋白在哺乳动物细胞中的运输功能。在目标2下，将进行经过验证的RNAi馈送筛选，以确定NEKL信令网络的组件和候选目标。此外，最近开发的一种强大的化学遗传-蛋白质组策略，得到了初步数据的良好支持，将被用于直接在体内鉴定NEKL的底物。后续研究将在SELECT VALIDATED 目标是阻止他们在窝点下游的贩运和细胞骨架组织中发挥作用。这种方法是创新的，因为其他研究小组还没有应用强大的正向遗传学方法来研究NEK激酶，也因为Neks在细胞内运输中的功能完全未知。此外，拟议的识别底物的化学遗传策略以前还没有应用于线虫或任何系统中的Neks的研究。这项拟议的研究意义重大，因为人类Nek广泛与人类疾病有关，并被认为是药物靶点，但它们在间期的作用尚不清楚，在大多数情况下，其生理底物也不为人所知。作为一个额外的好处，这些研究将导致表征以前未知但保守的细胞内转运和细胞外基质重塑的调节因子，这两者都与人类发育、健康和疾病高度相关。

 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.