LRRK2 regulates ArfGAP1 membrane localization, activity and neuronal integrity via phosphorylation within its lipid-sensing ALPS2 motif.

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IntroductionMutations 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.MethodsHere, we further explore the phosphorylation of ArfGAP1 by LRRK2 and its functional consequences.ResultsLRRK2 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.DiscussionOur 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.

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