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Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

The University of Tokyo

the university of tokyo

Involvement of the peripheral autonomic nervous system is a core feature of Lewy body (LB) diseases, including Parkinson disease (PD), PD with dementia, and dementia with LBs. To investigate the potential use of skin biopsy for the diagnosis of LB diseases, we assessed anti-phosphorylated alpha-synuclein immunoreactivity in peripheral nerves in samples of skin from the abdominal wall and flexor surface of the upper arm in 279 prospectively studied consecutively autopsied patients whose data were registered at the Brain Bank for Aging Research between 2002 and 2005. Positive immunoreactivity was demonstrated in the unmyelinated fibers of the dermis in 20 of 85 patients with LB pathology in the CNS and the adrenal glands, the latter representing a substitute for peripheral autonomic nervous system sympathetic ganglia; no reactivity was seen in 194 patients without CNS LB pathology. In 142 retrospectively studied patients autopsied from 1995 onward who had subclinical or clinical LB disease, the sensitivity of the positive skin immunoreactivity was 70% in PD and PD with dementia and 40% in dementia with LBs. Skin immunoreactivity was absent in cases of multiple-system atrophy, progressive nuclear palsy, and corticobasal degeneration. We demonstrate for the first time that the skin is involved and may be a highly specific and useful biopsy site for the pathological diagnosis of LB diseases.

Lewy body pathology involves cutaneous nerves.

α-Synuclein is causative for autosomal dominant familial Parkinson disease and dementia with Lewy bodies, and the phosphorylation of α-synuclein at residue Ser-129 is a key posttranslational modification detected in Parkinson disease/dementia with Lewy bodies lesions. However, the role of Ser-129 phosphorylation on the pathogenesis of Parkinson disease/dementia with Lewy bodies remains unclear. Here we investigated the neurotoxicity of Ser-129-substituted α-synuclein in the transgenic Caenorhabditis elegans (Tg worm) model of synucleinopathy. Tg worms pan-neuronally overexpressing nonphosphorylatable (S129A) α-synuclein showed severe defects including motor dysfunction, growth retardation, and synaptic abnormalities. In contrast, Tg worms expressing phosphorylation mimic (S129D) α-synuclein exhibited nearly normal phenotypes. Biochemical fractionation revealed that the level of membrane-bound α-synuclein was significantly increased in S129A-α-synuclein Tg worms, whereas S129D- as well as A30P-α-synuclein displayed lower membrane binding properties. Furthermore, A30P/S129A double mutant α-synuclein did not cause neuronal dysfunction and displayed low membrane binding property. In human neuroblastoma SH-SY5Y cells, localization of S129A-α-synuclein to membranes was significantly increased. Finally, gene expression profiling of S129A-Tg worms revealed a dramatic up-regulation of Daf-16/FOXO pathway genes, which likely act against the dysfunction caused by S129A-α-synuclein. These results imply a role of Ser-129 phosphorylation of α-synuclein in the attenuation of α-synuclein-induced neuronal dysfunction and downstream stress response by lowering the membrane binding property.

Phosphorylation of α-synuclein protein at Ser-129 reduces neuronal dysfunction by lowering its membrane binding property in Caenorhabditis elegans.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified in pedigrees of autosomal-dominant familial Parkinson's disease (PARK8). It has been shown that the kinase activity of LRRK2 is required for its neuronal toxicity, although how familial Parkinson mutations affect the function of LRRK2 has not been well characterized. In the present study, we systematically characterized the autophosphorylation of LRRK2 by phosphopeptide mapping and identified Thr1348, Thr1349, and Thr1357 as the major autophosphorylation sites. We found that the autophosphorylation at Thr1357 is downregulated by the Y1699C mutation, possibly through a conformational alteration of the ROC domain. We also found that I2020T mutant LRRK2 undergoes excessive autophosphorylation in cell lysates in vitro at a low concentration of ATP. These results highlight the differential effects of familial mutations in LRRK2 on its conformation and enzymatic properties.

Differential effects of familial parkinson mutations in LRRK2 revealed by a systematic analysis of autophosphorylation.

Leucine-rich repeat kinase 2 (LRRK2) is extensively phosphorylated in cells within a region amino-terminal to the leucine-rich repeat domain. Since phosphorylation in this region of LRRK2, including Ser910, Ser935, Ser955, and Ser973, is significantly downregulated upon treatment with inhibitors of LRRK2, it has been hypothesized that signaling pathways downstream of the kinase activity of LRRK2 are involved in regulating the phosphorylation of LRRK2, although the precise mechanism has remained unknown. Here we examined the effects of LRRK2 inhibitors on the phosphorylation state at Ser910, Ser935, and Ser955 in a series of kinase-inactive mutants of LRRK2. We found that the responses of LRRK2 to the inhibitors varied among mutants, in a manner not consistent with the above-mentioned hypothesis. Notably, one of the kinase-inactive mutants, T2035A LRRK2, underwent phosphorylation, as well as the inhibitor-induced dephosphorylation, at Ser910, Ser935, and Ser955, to a similar extent to those observed with wild-type LRRK2. These results suggest that the kinase activity of LRRK2 is not involved in the common mechanism of inhibitor-induced dephosphorylation of LRRK2.

Lack of correlation between the kinase activity of LRRK2 harboring kinase-modifying mutations and its phosphorylation at Ser910, 935, and Ser955.

Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1-2 min, markedly more rapidly than the Ser(935) and Ser(1292) biomarker sites that require 40-80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser(910) and Ser(935) and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.

Article	Year
Lewy body pathology involves cutaneous nerves. Journal of neuropathology and experimental neurology\| PubMed ID: 18800013	2008
Phosphorylation of α-synuclein protein at Ser-129 reduces neuronal dysfunction by lowering its membrane binding property in Caenorhabditis elegans. The Journal of biological chemistry\| PubMed ID: 22232559	2012
Differential effects of familial parkinson mutations in LRRK2 revealed by a systematic analysis of autophosphorylation. Biochemistry\| PubMed ID: 23924436	2013
Lack of correlation between the kinase activity of LRRK2 harboring kinase-modifying mutations and its phosphorylation at Ser910, 935, and Ser955. PloS one\| PubMed ID: 24836358	2014
Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors. The Biochemical journal\| PubMed ID: 27474410	2016

Genta Ito

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