University of Dundee, Scotland, UK.
Biochem J. 2010 Sep 15;430(3):405-13. doi: 10.1042/BJ20100784.
LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients. Since a common mutation that replaces Gly2019 with a serine residue enhances kinase catalytic activity, small-molecule LRRK2 inhibitors might have utility in treating Parkinson's disease. However, the effectiveness of inhibitors is difficult to assess, as no physiological substrates or downstream effectors have been identified that could be exploited to develop a robust cell-based assay. We recently established that LRRK2 bound 14-3-3 protein isoforms via its phosphorylation of Ser910 and Ser935. In the present study we show that treatment of Swiss 3T3 cells or lymphoblastoid cells derived from control or a Parkinson's disease patient harbouring a homozygous LRRK2(G2019S) mutation with two structurally unrelated inhibitors of LRRK2 (H-1152 or sunitinib) induced dephosphorylation of endogenous LRRK2 at Ser910 and Ser935, thereby disrupting 14-3-3 interaction. Our results suggest that H-1152 and sunitinib induce dephosphorylation of Ser910 and Ser935 by inhibiting LRRK2 kinase activity, as these compounds failed to induce significant dephosphorylation of a drug-resistant LRRK2(A2016T) mutant. Moreover, consistent with the finding that non-14-3-3-binding mutants of LRRK2 accumulated within discrete cytoplasmic pools resembling inclusion bodies, we observed that H-1152 causes LRRK2 to accumulate within inclusion bodies. These findings indicate that dephosphorylation of Ser910/Ser935, disruption of 14-3-3 binding and/or monitoring LRRK2 cytoplasmic localization can be used as an assay to assess the relative activity of LRRK2 inhibitors in vivo. These results will aid the elaboration and evaluation of LRRK2 inhibitors. They will also stimulate further research to understand how phosphorylation of Ser910 and Ser935 is controlled by LRRK2, and establish any relationship to development of Parkinson's disease.
LRRK2（富含亮氨酸重复蛋白激酶 2）在大量帕金森病患者中发生突变。由于取代甘氨酸 2019 的丝氨酸残基的常见突变增强了激酶的催化活性，小分子 LRRK2 抑制剂可能在治疗帕金森病方面具有应用价值。然而，由于尚未确定可以利用来开发强大的基于细胞的测定的生理底物或下游效应物，因此抑制剂的有效性难以评估。我们最近确定 LRRK2 通过其对 Ser910 和 Ser935 的磷酸化与 14-3-3 蛋白同工型结合。在本研究中，我们表明，用两种结构上无关的 LRRK2 抑制剂（H-1152 或舒尼替尼）处理瑞士 3T3 细胞或源自携带纯合 LRRK2（G2019S）突变的帕金森病患者的淋巴母细胞，可诱导内源性 LRRK2 在 Ser910 和 Ser935 处去磷酸化，从而破坏 14-3-3 相互作用。我们的结果表明，H-1152 和舒尼替尼通过抑制 LRRK2 激酶活性诱导 Ser910 和 Ser935 的去磷酸化，因为这些化合物未能诱导药物抗性 LRRK2（A2016T）突变体的显著去磷酸化。此外，与 LRRK2 的非 14-3-3 结合突变体在类似于包含体的离散细胞质池内积累的发现一致，我们观察到 H-1152 导致 LRRK2 在包含体内积累。这些发现表明 Ser910/Ser935 的去磷酸化、14-3-3 结合的破坏和/或监测 LRRK2 细胞质定位可用作评估体内 LRRK2 抑制剂相对活性的测定。这些结果将有助于 LRRK2 抑制剂的制定和评估。它们还将激发进一步的研究，以了解 Ser910 和 Ser935 的磷酸化如何受 LRRK2 控制，并确定与帕金森病发展的任何关系。
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