Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser ⁹¹⁰/Ser⁹³⁵, disruption of 14-3-3 binding and altered cytoplasmic localization

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 Ser ⁹¹⁰ and Ser⁹³⁵. In the present study we showthat 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 Ser ⁹³⁵, thereby disrupting 14-3-3 interaction. Our results suggest that H-1152 and sunitinib induce dephosphorylation of Ser⁹¹⁰ and Ser ⁹³⁵ 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 howphosphorylation of Ser⁹¹⁰ and Ser⁹³⁵ is controlled by LRRK2, and establish any relationship to development of Parkinson's disease.