Regulation of Leucine Rich Repeat Kinase 2 (LRRK2)

描述（由申请人提供）：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40%的病例。LRRK2突变也出现在前所未有数量的散发性PD患者中，暗示该蛋白存在于所有形式的PD中。虽然LRRK2功能障碍与PD发病机制之间的遗传联系是明确的，但对LRRK2的生理活性及其调控机制知之甚少。然而，LRRK2疾病突变的弱外显率确实表明，其他基因可能潜在地影响LRRK2的功能并在其调控中发挥作用。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对应物的几倍。此外，大部分（约75%）的LRRK2存在于静止的细胞质中，而膜部分则选择性地富集于LRRK2二聚体中。这与参与细胞内信号传导的其他蛋白质非常相似，其中各种激酶和gtp酶穿梭于膜之间，并可以通过二聚化激活。因此，我们假设LRRK2这些新发现的生化特性与自然界中用于调节激酶活性和细胞内信号传导的规范机制一致。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相关LRRK2突变体的生化性质。然而，这仍然是难以捉摸的，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。本应用程序的目标是优化新开发的LRRK2二聚化定量分析，并使用该工具揭示LRRK2调控的关键方面。具体来说，我们将1)开发LRRK2二聚化的蛋白质片段互补试验，2)鉴定完整细胞中LRRK2二聚化的细胞和遗传决定因素。这项工作不仅将告诉我们细胞内LRRK2信号通路，而且还将为未来研究几种致病性LRRK2突变的潜在机制提供新的工具。

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.