LRRK2 phosphorylates membrane-bound Rabs and is activated by GTP-bound Rab7L1 to promote recruitment to the trans-Golgi network

Abstract

Human genetic studies implicate LRRK2 and RAB7L1 in susceptibility to Parkinson disease (PD). These two genes function in the same pathway, as knockout of Rab7L1 results in phenotypes similar to LRRK2 knockout, and studies in cells and model organisms demonstrate LRRK2 and Rab7L1 interact in the endolysosomal system. Recently, a subset of Rab proteins have been identified as LRRK2 kinase substrates. Herein, we find that Rab8, Rab10, and Rab7L1 must be membrane and GTP-bound for LRRK2 phosphorylation. LRRK2 mutations that cause PD including R1441C, Y1699C, and G2019S all increase LRRK2 phosphorylation of Rab7L1 four-fold over wild-type LRRK2 in cells, resulting in the phosphorylation of nearly one-third the available Rab7L1 protein in cells. In contrast, the most common pathogenic LRRK2 mutation, G2019S, does not upregulate LRRK2-mediated phosphorylation of Rab8 or Rab10. LRRK2 interaction with membrane and GTP-bound Rab7L1, but not Rab8 or Rab10, results in the activation of LRRK2 autophosphorylation at the serine 1292 position, required for LRRK2 toxicity. Further, Rab7L1 controls the proportion of LRRK2 that is membrane-associated, and LRRK2 mutations enhance Rab7L1-mediated recruitment of LRRK2 to the trans-Golgi network. Interaction studies with the Rab8 and Rab10 GTPase-activating protein TBC1D4/AS160 demonstrate that LRRK2 phosphorylation may block membrane and GTP-bound Rab protein interaction with effectors. These results suggest reciprocal regulation between LRRK2 and Rab protein substrates, where Rab7L1-mediated upregulation of LRRK2 kinase activity results in the stabilization of membrane and GTP-bound Rab proteins that may be unable to interact with Rab effector proteins.

LRRK2 and Rab10 Coordinate Macropinocytosis to Mediate Immunological Responses in Phagocytes

“…Our previous experiments, consistent with others [12][13][14] , reveal that a proportion of membrane associated GTP-bound Rab10 protein in transfected cell lines is phosphorylated by LRRK2 in the switch II loop at residue T73, exquisitely dependent on LRRK2 kinase activity in these cells. Consistent with our previous results, immunofluorescence staining using a pT73-Rab10 specific antibody confirmed that pT73-Rab10 signal exclusively localized to vesicular structures, as opposed to total Rab10 signal that can be diffuse (cytoplasmic) and only partially localized to vesicles (Supporting data Figure 4).…”

“…LRRK2-mediated phosphorylation of Rab10 is strongly affected by all known pathogenic mutations as well as PD-risk factor genetic variants like G2385R, with Rab10 phosphorylation changing the affinity of different binding proteins to the Rab10 effector loops 12 . Our past work, suggests LRRK2 phosphorylation of Rab10 may prolong Rab10 in a GTP-bound state by inhibiting Rab10 interactions with GAP proteins 13 . While the specific role of Rab10 in cells linked to neurodegeneration is under investigation, recent work demonstrates Rab10 downregulates ciliogenesis in some types of cells and neurons in the brain 14 .…”

“…However, bath-application of cells (~20 minutes) with LRRK2 kinase inhibitor MLi2 completely ablated the pT73-Rab10-positive vesicle pool and decreased both Rab10 and Rab5-double positive vesicles to just ~5% of total Rab10 vesicles ( Figure 6 D, E). Our previous work in HEK-293 cells transfected with LRRK2 and Rab10 suggests LRRK2 phosphorylation of Rab10 may prolong Rab10 in a GTP-bound state by blocking Rab10-GAP interactions that occur near the phosphorylation site of the switch II domain 13 . Together, these results suggest that LRRK2 mediated phosphorylation stalls Rab10 on macropinosomes to promote macropinosome maturation, mimicking GTP-locked Rab10 albeit less dramatically.…”

“…LRRK2 is transiently recruited by GTP bound active Rab10 on macropinosomes and dissociates from Rab10 once it finished phosphorylating Rab10. LRRK2-mediated phosphorylation may block the interaction of Rab10 GAPs (GTPase-activating proteins) that would otherwise facilitate the GTPase activity of Rab10 and promote Rab10 dissociation from macropinosomes 13 . Without GAPs, Rab10 remained GTP bound and associated with macropinosomes.…”

Sequential screening nominates the Parkinson’s disease associated kinase LRRK2 as a regulator of Clathrin-mediated endocytosis

“…54 In cells, RAB29 has been shown to bind LRRK2 to increase TGN-localisation and kinase activity. 15,21,55 Our siRNA screen nominated AP2A1 as a strong modifier of LRRK2 recruitment and showed that depletion of AP2A1 in the presence of RAB29 caused a further increase of LRRK2 autophosphorylation. The mechanism by which this occurs is not yet known.…”