Ser ¹²⁹² Autophosphorylation Is an Indicator of LRRK2 Kinase Activity and Contributes to the Cellular Effects of PD Mutations

Abstract

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson's disease (PD). Although biochemical studies have shown that certain PD mutations confer elevated kinase activity in vitro on LRRK2, there are no methods available to directly monitor LRRK2 kinase activity in vivo. We demonstrate that LRRK2 autophosphorylation on Ser(1292) occurs in vivo and is enhanced by several familial PD mutations including N1437H, R1441G/C, G2019S, and I2020T. Combining two PD mutations together further increases Ser(1292) autophosphorylation. Mutation of Ser(1292) to alanine (S1292A) ameliorates the effects of LRRK2 PD mutations on neurite outgrowth in cultured rat embryonic primary neurons. Using cell-based and pharmacodynamic assays with phosphorylated Ser(1292) as the readout, we developed a brain-penetrating LRRK2 kinase inhibitor that blocks Ser(1292) autophosphorylation in vivo and attenuates the cellular consequences of LRRK2 PD mutations in vitro. These data suggest that Ser(1292) autophosphorylation may be a useful indicator of LRRK2 kinase activity in vivo and may contribute to the cellular effects of certain PD mutations.

Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3

“…In particular, we have shown that the LRRK2 mutants R1441H and S1444A, both of which prevent 14-3-3 binding to the ROC domain, had an ∼1.6 times enhanced LRRK2 kinase activity. This finding is in agreement with other studies in which enhanced kinase activity already was correlated with the PD-linked LRRK2 mutation R1441C (5,9). On the other hand, LRRK2 WT kinase activity is decreased in the presence of 14-3-3 and PKA in vitro, whereas LRRK2 S1444A (P-site) or R1441H (P-3 site) are unaffected.…”

“…Particularly for a single residue located within the ROC domain, three independent PDassociated mutations (R1441C, R1441G, and R1441H) have been found (4), whereas the kinase domain may harbor the most frequent pathogenic mutation, G2019S. Mutations at both these sites have been associated with enhanced kinase activity compared with wild type (5,6), suggesting that dysregulation of these enzymatic activities may contribute to PD pathogenesis.…”

Heterogeneity of Leucine-Rich Repeat Kinase 2 Mutations: Genetics, Mechanisms and Therapeutic Implications

“…Direct evaluation of GTPase and kinase activity of N1437H LRRK2 has not yet been reported, although this variant does cause increased GTP binding [58]. When overexpressed in HEK293 cells, N1437H LRRK2 has an increased phosphorylation at Ser1292 [105] and diminished phosphorylation of Ser935 and, hence, 14-3-3 binding [92]. The measured effects are similar to other ROC-COR domain mutations, supporting pathogenicity.…”

Development of Inducible Leucine-rich Repeat Kinase 2 (LRRK2) Cell Lines for Therapeutics Development in Parkinson's Disease

“…Although the exact biological function(s) of LRRK2 and its role in biochemical pathways are under investigation, several potential substrates have been identified, including LRRK2, Akt1, ezrin/radxin/moesin (ERM) proteins, β-tubulin, eukaryotic initiation factor 4E-binding protein 1, and mitogenactivated kinase 3, 4, 6, and 7 [13,[22][23][24][25][26][27][28][29][30][31][32][33][34]. The functional implications of the majority of these potential substrates with respect to LRRK2 patho-and physiological actions remain uncertain.…”

“…The functional implications of the majority of these potential substrates with respect to LRRK2 patho-and physiological actions remain uncertain. However, the work of Sheng et al [34] has directly implicated LRRK2 Ser1292 in pathogenic effects in cultured cells.…”