Regulation of Leucine Rich Repeat Kinase 2 (LRRK2)

描述(申请人提供)：多结构域激酶LRRK2内的错义突变是家族性帕金森病(PD)的最常见原因，在某些人群中占病例的40%。LRRK2突变也出现在前所未有数量的散发性帕金森病患者中，这表明该蛋白与所有形式的帕金森病有关。虽然LRRK2功能障碍与帕金森病发病机制之间的遗传联系是清楚的，但人们对LRRK2的生理活性或它是如何调节的知之甚少。然而，LRRK2疾病突变的微弱外显性确实表明，其他基因可能会有效地影响LRRK2的功能，并在其调节中发挥作用。最近的数据表明，与其他激酶一样，LRRK2可以在细胞内形成功能性二聚体。我们已经证明LRRK2二聚体的活性是其单体的几倍。此外，大多数(~75%)的LRRK2在静息状态下存在于细胞质中，而膜部分选择性地富含在LRRK2二聚体中。这与参与细胞内信号传递的其他蛋白质高度相似，在这些蛋白质中，各种激酶和GTP酶穿梭于细胞膜之间，并可通过二聚体激活。因此，我们假设这些新发现的LRRK2的生化特性与自然界中用于调节激酶活性和细胞内信号转导的典型机制是一致的。然而，这些过程对LRRK2的确切重要性尚不清楚。我们的长期目标是揭示PD连锁LRRK2突变体的生化性质。然而，这仍然难以捉摸，部分原因是我们对LRRK2生物学的了解不完全。此时，迫切需要对野生型LRRK2的生理功能和调控有更广泛的了解。这项应用的目标是优化一种新开发的LRRK2二聚化定量检测方法，并使用该工具来揭示LRRK2调控的关键方面。具体地说，我们将1)建立LRRK2二聚化的蛋白质片段互补分析，2)在完整的细胞中鉴定LRRK2二聚化的细胞和遗传决定因素。这项工作不仅将使我们了解细胞内LRRK2信号转导的途径，还将为在未来的研究中研究几种致病LRRK2突变的潜在机制提供新的工具。 与公共卫生相关：LRRK2激酶的突变是帕金森氏症最常见的遗传原因。这项应用的目标是优化一种新颖的、高通量的LRRK2生物学分析，以揭示细胞如何调控LRRK2功能。通过这个新的资源，以及对控制LRRK2活性的途径的更好的理解，我们希望对LRRK2的生理和病理功能获得有价值的见解。

DESCRIPTION (provided by applicant): Missense mutations within the multi-domain kinase, LRRK2, are the most common cause of familial Parkinson's disease (PD), accounting for up to 40% of cases in some populations. LRRK2 mutations also appear in an unprecedented number of sporadic PD patients, implicating this protein in all forms of PD. While the genetic links between LRRK2 dysfunction and PD pathogenesis are clear, less is known about the physiological activity of LRRK2 or how it is regulated. The weak penetrance of LRRK2 disease mutations, however, does suggest that other genes may potently influence LRRK2 function and play a role in its regulation. Recent data indicate that like other kinases, LRRK2 can form a functional dimer within the cell. We've shown that the LRRK2 dimer is several-fold more active than its monomeric counterpart. In addition, the majority (~75%) of total LRRK2 is found within the cytoplasm at rest, while the membrane fraction is selectively enriched in the LRRK2 dimer. This is highly reminiscent of other proteins involved in intracellular signaling, where various kinases and GTPases shuttle to and from the membrane, and can be activated through dimerization. Therefore, we hypothesize that these newly discovered biochemical properties of LRRK2 are consistent with canonical mechanisms used throughout nature to regulate kinase activity and intracellular signaling. However, the precise importance of these processes to LRRK2 are not known. Our long-term goal is to uncover the biochemical nature of PD-linked LRRK2 mutants. However, this has remained elusive, partly due to our incomplete understanding of LRRK2 biology. At this time, a broader understanding of the physiological function and regulation of wild-type LRRK2 is critically needed. The goal of this application is to optimize a newly developed, quantitative assay of LRRK2 dimerization, and use this tool to uncover critical aspects of LRRK2 regulation. Specifically, we will 1) develop a protein-fragment complementation assay of LRRK2 dimerization, and 2) identify cellular and genetic determinants of LRRK2 dimerization in the intact cell. This work will not only inform us as to the pathways involved in intracellular LRRK2 signaling, but also provide novel tools to examine the underlying mechanisms of several pathogenic LRRK2 mutations in future studies. PUBLIC HEALTH RELEVANCE: Mutations in the LRRK2 kinase are the most common genetic cause of Parkinson's disease. The goal of this application is to optimize a novel, high-throughput assay of LRRK2 biology to uncover how cells regulate LRRK2 function. Through this new resource, and an improved understanding of the pathways controlling LRRK2 activity, we hope to gain valuable insights into the physiological and pathological functions of LRRK2.