Abstract
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.
Highlights
Autosomal dominant missense mutations that hyperactivate LRRK2 are one of the most common causes of familial Parkinson’s disease (PD) [1,2,3,4]
The LRRK2[R1441C/G] mutants are more readily activated by Rab29, which could explain why this mutation elevates LRRK2 activity
We found that knock-out of Rab29 does not impact elevated Rab10 or Rab12 phosphorylation observed in the LRRK2[R1441C] knock-in mouse embryonic fibroblast (MEF) or mouse tissues
Summary
Autosomal dominant missense mutations that hyperactivate LRRK2 (leucine-rich repeat kinase 2) are one of the most common causes of familial Parkinson’s disease (PD) [1,2,3,4]. LRRK2 is a large, multi-functional protein kinase that encodes two central catalytic regions, a Roc-type GTPase domain adjacent to a COR (C-terminal of Roc) domain, which is followed by a serine/threonine protein kinase domain. These enzymatic regions are surrounded by several domains, including N-terminal armadillo and ankyrin domains, leucine-rich repeats and a C-terminal WD-40 repeat [5]. The mutations within the GTPase domain do not directly activate LRRK2 activity in vitro, but enhance interaction with Rab located at the Golgi [14,15]. LRRK2 protein kinase inhibitors are currently in early stage clinical trials for LRRK2-driven PD [24,25]
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