MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee, U.K.
Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, U.S.A.
Biochem J. 2022 Sep 16;479(17):1759-1783. doi: 10.1042/BCJ20220161.
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.
LRRK2 激酶结构域中增强激酶活性的突变会导致帕金森病（PD），目前正在临床试验中测试降低 LRRK2 激酶活性的疗法。已经报道了许多具有未知临床意义的罕见变异，但绝大多数变异如何影响 LRRK2 功能尚不清楚。在这里，我们研究了 100 种与 PD 相关的 LRRK2 变异体，包括先前描述的致病性突变。我们确定了 23 种可强烈刺激激酶活性的 LRRK2 变异体，包括位于 N 端非催化区的变异体（ARM（E334K、A419V）、ANK（R767H）、LRR（R1067Q、R1325Q）），以及预测会破坏 ROC：CORB 界面稳定性的变异体（ROC（A1442P、V1447M）、CORA（R1628P）、CORB（S1761R、L1795F））和 COR：COR 二聚体界面（CORB（R1728H/L））。大多数激活变异体降低 LRRK2 生物标志物位点磷酸化（pSer935/pSer955/pSer973），这与活性激酶构象阻止其磷酸化的观点一致。我们得出结论，与生化激酶测定相比，通过 LRRK2 依赖性 Rab10 底物磷酸化的细胞测定评估变体对激酶活性的影响效果最佳，因为只有少数激活变体（CORB（Y1699C、R1728H/L、S1761R）和激酶（G2019S、I2020T、T2031S））增强了免疫沉淀 LRRK2 的体外激酶活性。包括几种激活 LRRK2 并与 PD 相关的变异体在内的 12 种变异体，在 I 型激酶抑制剂存在的情况下抑制微管结合（ARM（M712V）、LRR（R1320S）、ROC（A1442P、K1468E、S1508R）、CORA（A1589S）、CORB（Y1699C、R1728H/L）和 WD40（R2143M、S2350I、G2385R））。我们的发现将激发人们更好地理解变异体影响生物学的机制，并为正在进行和未来的 LRRK2 抑制剂临床试验中变异体携带者的纳入或排除提供依据。
