A QUICK Screen for Lrrk2 Interaction Partners - Leucine-rich Repeat Kinase 2 is Involved in Actin Cytoskeleton Dynamics
A QUICK Screen for Lrrk2 Interaction Partners - Leucine-rich Repeat Kinase 2 is Involved in Actin Cytoskeleton Dynamics
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DOI：
10.1074/mcp.m110.001172
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发表时间：
2011-01-01
影响因子：
7
通讯作者：
Ueffing, Marius
中科院分区：
文献类型：
--
作者：
Meixner, Andrea;Boldt, Karsten;Ueffing, Marius
人类富含亮氨酸重复蛋白2(LRRK2)是一种功能未知的蛋白质，它的突变与中脑多巴胺能神经元退化引起的帕金森氏症有关。该蛋白包括几个结构域，包括一个GTP酶和一个激活域，两者都受到几种致病突变的影响。为了阐明化学计量约束下内源性LRRK2的分子相互作用网络，我们在NIH3T3细胞中应用了QUICK(定量免疫沉淀结合基因敲除)。已鉴定的相互作用组揭示了肌动蛋白的异构体以及参与肌动蛋白细丝组装、组织、重排和维持的肌动蛋白相关蛋白，提示LRRK2的生物学功能与细胞骨架动力学有关。事实上，我们证明了LRRK2从头与F-肌动蛋白结合，以及它在体外调节其组装的能力。当在完整的细胞中进行测试时，LRRK2的敲除会导致NIH3T3细胞的形态变化。在发育的多巴胺能中脑初级神经元中，LRRK2基因敲除导致突起过程缩短，表明LRRK2在多巴胺能神经元的细胞骨架组织和动力学中具有生理作用。因此，我们的结果表明，LRRK2的分子相互作用以及生理功能与基于肌动蛋白的细胞骨架的组织密切相关，肌动蛋白是神经元发育和神经元功能的关键特征。分子与细胞蛋白质组学10：10.1074/mcp.M110.001172，1-17,2011年。
Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.001172, 1-17, 2011.