Impact of 100 LRRK2 variants linked to Parkinson's disease on kinase activity and microtubule binding.
Impact of 100 LRRK2 variants linked to Parkinson's disease on kinase activity and microtubule binding.
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DOI：
10.1042/bcj20220161
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发表时间：
2022-09-16
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增强富亮氨酸重复激酶2 （LRRK2）激酶活性的突变导致帕金森病（PD），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，但绝大多数对LRRK2功能的影响尚不清楚。在这里，我们研究了100个与PD相关的LRRK2变异，包括先前描述的致病突变。我们发现了23个强有力地刺激激酶活性的LRRK2变体，包括n端非催化区域内的变体(ARM (E334K, A419V), ANK (R767H), LRR (R1067Q, R1325Q))，以及预测会破坏ROC:CORB界面(ROC (A1442P, V1447M), CORA （R1628P) CORB (S1761R, L1795F)）和COR:COR二聚体界面（CORB (R1728H/L)）的变体。大多数激活变异降低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。我们得出结论，变体对激酶活性的影响最好通过LRRK2依赖性Rab10底物磷酸化的细胞试验来评估，与生化激酶试验相比，只有少数激活变体（CORB （Y1699C, R1728H/L, S1761R）和激酶（G2019S, I2020T, T2031S））增强免疫沉淀的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(ARM (M712V), LRR (R1320S), ROC (A1442P, K1468E, S1508R), CORA （A1589S), CORB （Y1699C, R1728H/L）和WD40 (R2143M, S2350I, G2385R)）存在时抑制微管关联。我们的研究结果将有助于更好地理解变异影响生物学的机制，并为正在进行和未来的LRRK2抑制剂临床试验中变异载体的包含或排除提供依据。
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.