Dissecting the effects of GTPase and kinase domain mutations on LRRK2 endosomal localization and activity

Abstract

Parkinson's disease-causing LRRK2 mutations lead to varying degrees of Rab GTPase hyperphosphorylation. Puzzlingly, LRRK2 GTPase-inactivating mutations--which do not affect intrinsic kinase activity--lead to higher levels of cellular Rab phosphorylation than kinase-activating mutations. Here, we investigated whether mutation-dependent differences in LRRK2 cellular localization could explain this discrepancy. We discovered that blocking endosomal maturation leads to the rapid formation of mutant LRRK2+ endosomes on which LRRK2 phosphorylates substrate Rabs. LRRK2+ endosomes are maintained through positive feedback, which mutually reinforces membrane localization of LRRK2 and phosphorylated Rab substrates. Furthermore, across a panel of mutants, cells expressing GTPase-inactivating mutants formed strikingly more LRRK2+ endosomes than cells expressing kinase-activating mutants, resulting in higher total cellular levels of phosphorylated Rabs. Our study suggests that an increased probability of LRRK2 GTPase-inactivating mutants to be retained on intracellular membranes over the kinase-activating mutants leads to higher substrate phosphorylation.

Insights into the cellular consequences of LRRK2-mediated Rab protein phosphorylation

“…Thus, additional mechanisms related to Rab dephosphorylation and/or lysosomal positioning may contribute to the selective accumulation of only certain phospho-Rab proteins on distinct membranes. Finally, apart from either a Rab29-mediated or a cooperative phospho-Rab-mediated recruitment, other mechanisms related to phospholipid composition [ 91 ] and/or intraorganellar calcium [ 85 ] may contribute to the selective recruitment and activation of LRRK2 at distinct endolysosomal membranes. Since membrane recruitment results in LRRK2 activation and substrate phosphorylation, further work is warranted to understand the precise cellular triggers for LRRK2 activation at distinct intracellular membranes.…”