Laboratory for Biomolecular Modelling, Division of Biochemistry, Molecular and Structural Biology, Department of Chemistry, KU Leuven Leuven, Belgium.
Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven Leuven, Belgium.
Front Mol Neurosci. 2014 Jun 3;7:51. doi: 10.3389/fnmol.2014.00051. eCollection 2014.
Leucine-rich repeat kinase 2 (LRRK2) is a complex, multidomain protein which is considered a valuable target for potential disease-modifying therapeutic strategies for Parkinson's disease (PD). In mammalian cells and brain, LRRK2 is phosphorylated and treatment of cells with inhibitors of LRRK2 kinase activity can induce LRRK2 dephosphorylation at a cluster of serines including Ser910/935/955/973. It has been suggested that phosphorylation levels at these sites reflect LRRK2 kinase activity, however kinase-dead variants of LRRK2 or kinase activating variants do not display altered Ser935 phosphorylation levels compared to wild type. Furthermore, Ser910/935/955/973 are not autophosphorylation sites, therefore, it is unclear if inhibitor induced dephosphorylation depends on the activity of compounds on LRRK2 or on yet to be identified upstream kinases. Here we used a panel of 160 ATP competitive and cell permeable kinase inhibitors directed against all branches of the kinome and tested their activity on LRRK2 in vitro using a peptide-substrate-based kinase assay. In neuronal SH-SY5Y cells overexpressing LRRK2 we used compound-induced dephosphorylation of Ser935 as readout. In silico docking of selected compounds was performed using a modeled LRRK2 kinase structure. Receiver operating characteristic plots demonstrated that the obtained docking scores to the LRRK2 ATP binding site correlated with in vitro and cellular compound activity. We also found that in vitro potency showed a high degree of correlation to cellular compound induced LRRK2 dephosphorylation activity across multiple compound classes. Therefore, acute LRRK2 dephosphorylation at Ser935 in inhibitor treated cells involves a strong component of inhibitor activity on LRRK2 itself, without excluding a role for upstream kinases. Understanding the regulation of LRRK2 phosphorylation by kinase inhibitors aids our understanding of LRRK2 signaling and may lead to development of new classes of LRRK2 kinase inhibitors.
富含亮氨酸重复激酶 2（LRRK2）是一种复杂的、多结构域蛋白，被认为是帕金森病（PD）潜在疾病修饰治疗策略的有价值的靶点。在哺乳动物细胞和大脑中，LRRK2 被磷酸化，用 LRRK2 激酶活性抑制剂处理细胞可以诱导 LRRK2 在包括 Ser910/935/955/973 在内的一组丝氨酸上脱磷酸化。有人认为这些位点的磷酸化水平反映了 LRRK2 激酶活性，但是 LRRK2 的激酶失活变体或激酶激活变体与野生型相比，Ser935 磷酸化水平没有改变。此外，Ser910/935/955/973 不是自身磷酸化位点，因此，尚不清楚抑制剂诱导的脱磷酸化是否依赖于化合物对 LRRK2 的活性或尚未确定的上游激酶。在这里，我们使用了针对激酶组所有分支的 160 种 ATP 竞争性和细胞渗透性激酶抑制剂，使用基于肽底物的激酶测定法在体外测试了它们对 LRRK2 的活性。在过表达 LRRK2 的神经元 SH-SY5Y 细胞中，我们使用化合物诱导的 Ser935 脱磷酸化作为读出。使用建模的 LRRK2 激酶结构对选定化合物进行了计算机对接。接收者操作特性图表明，获得的与 LRRK2 ATP 结合位点的对接分数与体外和细胞内化合物活性相关。我们还发现，在多个化合物类别中，体外效力与细胞内化合物诱导的 LRRK2 脱磷酸化活性具有高度相关性。因此，在抑制剂处理的细胞中，LRRK2 第 935 位丝氨酸的急性脱磷酸化涉及抑制剂对 LRRK2 本身的强烈活性成分，而不排除上游激酶的作用。了解激酶抑制剂对 LRRK2 磷酸化的调节有助于我们了解 LRRK2 信号转导，并可能导致开发新的 LRRK2 激酶抑制剂。
