Mutations that enhance LRRK2 protein kinase activity cause inherited Parkinson's disease. LRRK2 phosphorylates a group of Rab GTPase proteins, including Rab10 and Rab12, within the effector-binding switch-II motif. Previous work has indicated that the PARK16 locus, which harbors the gene encoding for Rab29, is involved in Parkinson's, and that Rab29 operates in a common pathway with LRRK2. Co-expression of Rab29 and LRRK2 stimulates LRRK2 activity by recruiting LRRK2 to the surface of the trans Golgi network. Here, we report that knock-out of Rab29 does not influence endogenous LRRK2 activity, based on the assessment of Rab10 and Rab12 phosphorylation, in wild-type LRRK2, LRRK2[R1441C] or VPS35[D620N] knock-in mouse tissues and primary cell lines, including brain extracts and embryonic fibroblasts. We find that in brain extracts, Rab12 phosphorylation is more robustly impacted by LRRK2 inhibitors and pathogenic mutations than Rab10 phosphorylation. Transgenic overexpression of Rab29 in a mouse model was also insufficient to stimulate basal LRRK2 activity. We observed that stimulation of Rab10 and Rab12 phosphorylation induced by agents that stress the endolysosomal system (nigericin, monensin, chloroquine and LLOMe) is suppressed by LRRK2 inhibitors but not blocked in Rab29 deficient cells. From the agents tested, nigericin induced the greatest increase in Rab10 and Rab12 phosphorylation (5 to 9-fold). Our findings indicate that basal, pathogenic, as well as nigericin and monensin stimulated LRRK2 pathway activity is not controlled by Rab29. Further work is required to establish how LRRK2 activity is regulated, and whether other Rab proteins can control LRRK2 by targeting it to diverse membranes.

中文翻译：

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内源性 Rab29 不会影响基础或刺激的 LRRK2 通路活性

增强 LRRK2 蛋白激酶活性的突变会导致遗传性帕金森病。LRRK2 磷酸化效应器结合开关 II 基序内的一组 Rab GTPase 蛋白，包括 Rab10 和 Rab12。先前的研究表明，含有编码 Rab29 的基因的 PARK16 基因座与帕金森病有关，并且 Rab29 与 LRRK2 的运作途径相同。Rab29 和 LRRK2 的共表达通过将 LRRK2 招募到反式高尔基体网络表面来刺激 LRRK2 活性。在这里，我们报告，基于野生型 LRRK2、LRRK2[R1441C] 或 VPS35[D620N] 敲入小鼠组织和原代细胞中 Rab10 和 Rab12 磷酸化的评估，敲除 Rab29 不会影响内源 LRRK2 活性系，包括脑提取物和胚胎成纤维细胞。我们发现，在大脑提取物中，Rab12 磷酸化比 Rab10 磷酸化更容易受到 LRRK2 抑制剂和致病性突变的影响。Rab29 在小鼠模型中的转基因过表达也不足以刺激基础 LRRK2 活性。我们观察到，内溶酶体系统应激剂（尼日利亚菌素、莫能菌素、氯喹和 LLOMe）诱导的 Rab10 和 Rab12 磷酸化刺激被 LRRK2 抑制剂抑制，但在 Rab29 缺陷细胞中未被阻断。从测试的试剂来看，尼日利亚菌素诱导 Rab10 和 Rab12 磷酸化的最大增加（5 至 9 倍）。我们的研究结果表明，基础、致病性以及尼日利亚菌素和莫能菌素刺激的 LRRK2 通路活性不受 Rab29 控制。需要进一步的工作来确定 LRRK2 活性如何受到调节，以及其他 Rab 蛋白是否可以通过将 LRRK2 靶向不同的膜来控制 LRRK2。