Abstract
Mutations that activate the LRRK2 (leucine-rich repeat protein kinase 2) protein kinase predispose to Parkinson's disease, suggesting that LRRK2 inhibitors might have therapeutic benefit. Recent work has revealed that LRRK2 phosphorylates a subgroup of 14 Rab proteins, including Rab10, at a specific residue located at the centre of its effector-binding switch-II motif. In the present study, we analyse the selectivity and sensitivity of polyclonal and monoclonal phospho-specific antibodies raised against nine different LRRK2-phosphorylated Rab proteins (Rab3A/3B/3C/3D, Rab5A/5B/5C, Rab8A/8B, Rab10, Rab12, Rab29[T71], Rab29[S72], Rab35 and Rab43). We identify rabbit monoclonal phospho-specific antibodies (MJFF-pRAB10) that are exquisitely selective for LRRK2-phosphorylated Rab10, detecting endogenous phosphorylated Rab10 in all analysed cell lines and tissues, including human brain cingulate cortex. We demonstrate that the MJFF-pRAB10 antibodies can be deployed to assess enhanced Rab10 phosphorylation resulting from pathogenic (R1441C/G or G2019S) LRRK2 knock-in mutations as well as the impact of LRRK2 inhibitor treatment. We also identify rabbit monoclonal antibodies displaying broad specificity (MJFF-pRAB8) that can be utilised to assess LRRK2-controlled phosphorylation of a range of endogenous Rab proteins, including Rab8A, Rab10 and Rab35. The antibodies described in the present study will help with the assessment of LRRK2 activity and examination of which Rab proteins are phosphorylated in vivo. These antibodies could also be used to assess the impact of LRRK2 inhibitors in future clinical trials.
Highlights
Occurring autosomal-dominant missense mutations within the leucine-rich repeat protein kinase 2 (LRRK2) protein are linked to Parkinson’s disease [1,2]
Rab29 is unique, as in addition to Ser72 that lies at the equivalent position to other LRRK2 Rab protein phosphorylation sites, possesses an adjacent Thr71 residue that is phosphorylated by LRRK2 [12]
Six of the Rab protein LRRK2 phosphorylation sites are on threonine residues (Rab3, Rab8, Rab10, Rab29[Thr71], Rab35 and Rab43) and three on serine residues (Rab5, Rab12 and Rab29 [Ser72]), which could influence the selectivity of the phospho-specific antibodies (Figure 1)
Summary
Occurring autosomal-dominant missense mutations within the LRRK2 (leucine-rich repeat protein kinase 2) protein are linked to Parkinson’s disease [1,2]. LRRK2 is a large protein containing numerous domains including a ROC/COR-type GTPase and a serine/threonine protein kinase [4]. The most frequent pathogenic mutation (G2019S) is located within the kinase domain and directly enhances kinase activity, suggesting that LRRK2 inhibitors might offer therapeutic benefit for preventing and treating Parkinson’s disease [5,6,7,8]. All clear-cut LRRK2 pathogenic mutations tested, including the G2019S mutant, as well as the R1441G/C and Y1699C mutations located within the ROC/COR GTPase domain, markedly elevate Rab phosphorylation in cell lines (HEK293 and mouse embryonic fibroblasts) as well as mouse tissues (the brain, spleen, lung and kidney) [10,11]
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