Abstract
LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients. Since a common mutation that replaces Gly2019 with a serine residue enhances kinase catalytic activity, small-molecule LRRK2 inhibitors might have utility in treating Parkinson's disease. However, the effectiveness of inhibitors is difficult to assess, as no physiological substrates or downstream effectors have been identified that could be exploited to develop a robust cell-based assay. We recently established that LRRK2 bound 14-3-3 protein isoforms via its phosphorylation of Ser910 and Ser935. In the present study we show that treatment of Swiss 3T3 cells or lymphoblastoid cells derived from control or a Parkinson's disease patient harbouring a homozygous LRRK2(G2019S) mutation with two structurally unrelated inhibitors of LRRK2 (H-1152 or sunitinib) induced dephosphorylation of endogenous LRRK2 at Ser910 and Ser935, thereby disrupting 14-3-3 interaction. Our results suggest that H-1152 and sunitinib induce dephosphorylation of Ser910 and Ser935 by inhibiting LRRK2 kinase activity, as these compounds failed to induce significant dephosphorylation of a drug-resistant LRRK2(A2016T) mutant. Moreover, consistent with the finding that non-14-3-3-binding mutants of LRRK2 accumulated within discrete cytoplasmic pools resembling inclusion bodies, we observed that H-1152 causes LRRK2 to accumulate within inclusion bodies. These findings indicate that dephosphorylation of Ser910/Ser935, disruption of 14-3-3 binding and/or monitoring LRRK2 cytoplasmic localization can be used as an assay to assess the relative activity of LRRK2 inhibitors in vivo. These results will aid the elaboration and evaluation of LRRK2 inhibitors. They will also stimulate further research to understand how phosphorylation of Ser910 and Ser935 is controlled by LRRK2, and establish any relationship to development of Parkinson's disease.
Highlights
Autosomal dominant missense mutations within the gene encoding leucine-rich repeat protein kinase 2 (LRRK2) predispose humans to Parkinson’s disease [1,2]
To investigate how inhibition of LRRK2 protein kinase activity affects on Ser910/Ser935 phosphorylation, we initially treated Swiss 3T3 cells with increasing amounts of the LRRK2 inhibitors H-1152 (Figure 1A) or sunitinib (Figure 1C)
We found that 10–30 μM H-1152 or 3–10 μM sunitinib induced almost complete dephosphorylation of Ser910 and Ser935, resulting in a loss of 14-3-3 binding
Summary
Autosomal dominant missense mutations within the gene encoding LRRK2 (leucine-rich repeat protein kinase 2) predispose humans to Parkinson’s disease [1,2]. Patients with LRRK2 mutations generally develop Parkinson’s disease with clinical appearance and symptoms indistinguishable from idiopathic disease at around 60–70 years of age [3]. Mutations in LRRK2 account for 4 % of familial Parkinson’s disease, and are observed in 1 % of sporadic Parkinson’s disease patients [3]. Two other mutations (R1728H and T2031S) increase LRRK2 protein kinase activity [6]. These observations indicate that inhibitors of LRRK2 may have utility for the treatment of Parkinson’s disease
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