Regulation of Leucine Rich Repeat Kinase 2 (LRRK2)

描述（由申请人提供）：多结构域激酶LRRK 2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%。LRRK 2突变也出现在前所未有数量的散发性PD患者中，表明这种蛋白质存在于所有形式的PD中。虽然LRRK 2功能障碍和PD发病机制之间的遗传联系是明确的，但对LRRK 2的生理活性或如何调节知之甚少。然而，LRRK 2疾病突变的弱阳性率确实表明其他基因可能有力地影响LRRK 2功能并在其调节中发挥作用。最近的数据表明，像其他激酶一样，LRRK 2可以在细胞内形成功能性二聚体。我们已经证明LRRK 2二聚体比其单体对应物活性高几倍。此外，大部分（~75%）的总LRRK 2在静止时存在于细胞质中，而膜部分选择性地富集LRRK 2二聚体。这让人联想到参与细胞内信号传导的其他蛋白质，其中各种激酶和GTP酶往返于膜，并且可以通过二聚化被激活。因此，我们假设这些新发现的LRRK 2的生化特性与整个自然界用于调节激酶活性和细胞内信号传导的经典机制一致。然而，这些过程对LRRK 2的确切重要性尚不清楚。我们的长期目标是揭示PD连锁LRRK 2突变体的生物化学性质。然而，这仍然是难以捉摸的，部分原因是我们对LRRK 2生物学的不完全理解。目前，迫切需要对野生型LRRK 2的生理功能和调控有更广泛的了解。本申请的目标是优化新开发的LRRK 2二聚化定量测定，并使用该工具揭示LRRK 2调控的关键方面。具体而言，我们将1）开发LRRK 2二聚化的蛋白片段互补测定，2）鉴定完整细胞中LRRK 2二聚化的细胞和遗传决定因素。这项工作不仅将告知我们参与细胞内LRRK 2信号传导的途径，而且还提供了新的工具，以检查未来研究中几种致病性LRRK 2突变的潜在机制。 公共卫生相关性：LRRK 2激酶的突变是帕金森病最常见的遗传原因。该应用的目标是优化LRRK 2生物学的新型高通量测定，以揭示细胞如何调节LRRK 2功能。通过这一新的资源，以及对控制LRRK 2活性的途径的更好理解，我们希望获得对LRRK 2的生理和病理功能的有价值的见解。

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.