Genome-wide screen reveals Rab12 GTPase as a critical activator of pathogenic LRRK2 kinase

Abstract

Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2 dependent and PPM1H phosphatase reversible; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. AlphaFold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain and we show that residues predicted to be essential for Rab12 interaction at this site influence overall phosphoRab levels in a manner distinct from Rab29 activation of LRRK2. Our data support a model in which Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.

Biophysical analysis reveals autophosphorylation as an important negative regulator of LRRK2 dimerization

“…2C. In agreement with previous studies, multiple Rab binding sites are detected in the Armadillo domain (Dhekne et al, 2023; Vides et al, 2022). However, district contact patterns with the enzymatic core (Roc-COR-Kinase) of LRRK2 have also been detected.…”

A STING-CASM-GABARAP Pathway Activates LRRK2 at Lysosomes

“…In order define specific cellular machinery that is required for CASM-mediated LRRK2 activation, we performed a targeted siRNA screen focusing on the ATG8-related proteins that are lipidated as part of the CASM pathway. As expected, LRRK2 activity (measured by Rab10 phosphorylation) was reduced by the knockdown of LRRK2 and Rab10 itself as well as by knockdown of Rab12, a recently identified regulator of LRRK2 activity (Dhekne et al, 2023;Wang et al, 2023a)(Fig. 5A).…”

“…Rab GTPases also act upstream of LRRK2 to promote its membrane recruitment and activation (Dhekne et al, 2023;Eguchi et al, 2018;Gomez et al, 2019;Lian et al, 2023;Pfeffer, 2022;Purlyte et al, 2018;Unapanta et al, 2023;Vides et al, 2022;Wang et al, 2023a). However, although Rab GTPases promote LRRK2 kinase activity in specific contexts, recent knockout mouse studies revealed the persistence of partial or even full LRRK2 kinase activity in multiple tissues even after depletion of key Rab proteins implicated in LRRK2 activation (Dhekne et al, 2023;Kalogeropulou et al, 2020). These results suggest the existence of additional factors that contribute to LRRK2 activation.…”

Rab29-dependent asymmetrical activation of leucine-rich repeat kinase 2

“…Lastly, LRRK2 activation is clearly a complex process and could be achieved by other mechanisms, such as lipid oxidation, Rab12, and microtubule-based filamentation (11,12,(55)(56)(57)(58). This study focuses on Rab29-induced LRRK2 activation and provides a framework for interpreting disease mutations in the context of kinase activation.…”