Mutations enhancing the kinase activity of leucine-rich repeat kinase-2 (LRRK2) cause Parkinson's disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions (ARM (E334K, A419V), ANK (R767H), LRR (R1067Q, R1325Q)), as well as variants predicted to destabilize the ROC:CORB interface (ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)) and COR:COR dimer interface (CORB (R1728H/L)). Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation, compared with a biochemical kinase assay, as only a minority of activating variants (CORB (Y1699C, R1728H/L, S1761R) and kinase (G2019S, I2020T, T2031S)), enhance in vitro kinase activity of immunoprecipitated LRRK2. Twelve variants including several that activate LRRK2 and have been linked to PD, suppress microtubule association in the presence of a Type I kinase inhibitor (ARM (M712V), LRR (R1320S), ROC (A1442P, K1468E, S1508R), CORA (A1589S), CORB (Y1699C, R1728H/L) and WD40 (R2143M, S2350I, G2385R)). Our findings will stimulate work to better understand the mechanisms by which variants impact biology and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 inhibitor clinical trials.

中文翻译：

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与帕金森病相关的 100 个 LRRK2 变异对激酶活性和微管结合的影响


增强富含亮氨酸重复激酶 2 (LRRK2) 激酶活性的突变会导致帕金森病 (PD)，而降低 LRRK2 激酶活性的疗法正在临床试验中进行测试。已经报道了许多临床意义未知的罕见变异，但绝大多数如何影响 LRRK2 功能尚不清楚。在这里，我们研究了 100 个与 PD 相关的 LRRK2 变异，包括之前描述的致病突变。我们鉴定了 23 个强烈刺激激酶活性的 LRRK2 变体，包括 N 端非催化区域内的变体（ARM (E334K, A419V)、ANK (R767H)、LRR (R1067Q, R1325Q)），以及预计会破坏稳定性的变体ROC:CORB 接口（ROC (A1442P、V1447M)、CORA (R1628P) CORB (S1761R、L1795F)）和 COR:COR 二聚体接口（CORB (R1728H/L)）。大多数激活变体都会降低 LRRK2 生物标志物位点磷酸化 (pSer935/pSer955/pSer973)，这与活性激酶构象阻断其磷酸化的观点一致。我们的结论是，与生化激酶测定相比，通过部署 LRRK2 依赖性 Rab10 底物磷酸化的细胞测定，可以最好地评估变体对激酶活性的影响，因为只有少数激活变体 (CORB (Y1699C、R1728H/L、S1761R) ) 和激酶 (G2019S、I2020T、T2031S))，增强免疫沉淀 LRRK2 的体外激酶活性。 12 种变体，包括几种激活 LRRK2 并与 PD 相关的变体，在 I 型激酶抑制剂存在的情况下抑制微管关联（ARM (M712V)、LRR (R1320S)、ROC (A1442P、K1468E、S1508R)、CORA (A1589S) 、CORB（Y1699C、R1728H/L）和 WD40（R2143M、S2350I、G2385R））。 我们的研究结果将促进人们更好地了解变异影响生物学的机制，并为正在进行和未来的 LRRK2 抑制剂临床试验中包含或排除变异载体提供依据。