LRRK2 regulates ArfGAP1 membrane localization, activity and neuronal integrity via phosphorylation within its...

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing a Roc GTPase domain and a serine/threonine-directed protein kinase domain, with PD-linked mutations known to enhance LRRK2 kinase activity and neuronal toxicity. Our previous studies identified the Golgi protein, ADP-Ribosylation Factor GTPase-Activating Protein 1 (ArfGAP1), as a novel modifier of LRRK2-induced cellular toxicity, where it can serve as a GAP-like protein and a robust kinase substrate of LRRK2.

Here, we further explore the phosphorylation of ArfGAP1 by LRRK2 and its functional consequences.

LRRK2 mediates the robust phosphorylation of ArfGAP1 in vitro within its lipid-sensing ALPS2 motif at residues Ser284, Thr291, and Thr292. We mutated these three candidate phosphorylation sites, either alone or combined, to create hydrophobic phospho-null or charged phospho-mimicking versions of ArfGAP1. We find that modulating ArfGAP1 phosphorylation at these sites impairs its normal capacity to induce Golgi fragmentation upon overexpression in neural cells. Blocking phosphorylation impairs ArfGAP1-induced neurite outgrowth inhibition in primary neurons and protects against the pathogenic effects of PD-linked G2019S LRRK2. ArfGAP1 interactome analysis in neural cells identifies 114 putative interacting proteins with a proportion of these localized to mitochondria, including the outer membrane proteins Voltage-Dependent Anion Channel (VDAC) 1-3. An ArfGAP1 triple phospho-mimic mutant displays an increased interaction with mitochondrial VDACs owing to the redistribution of ArfGAP1 from the cis-Golgi to the cytoplasm. Mimicking ArfGAP1 phosphorylation also blocks the formation of Golgi-derived vesicles following mild ER stress.

Our data provides evidence for a complex functional interaction between LRRK2 and ArfGAP1 that serves to regulate ArfGAP1 subcellular localization, protein interactions, activity and neuronal integrity via LRRK2-mediated phosphorylation of its membrane-binding ALPS2 motif. Our findings support additional validation of ArfGAP1 as a putative therapeutic target for modulating LRRK2-linked PD.