G2385R and I2020T Mutations Increase LRRK2 GTPase Activity

The LRRK2 mutation is a major causal mutation in familial Parkinson’s disease. Although LRRK2 contains functional GTPase and kinase domains and their activities are altered by pathogenic mutations, most studies focused on LRRK2 kinase activity because the most prevalent mutant, G2019S, enhances kinase activity. However, the G2019S mutation is extremely rare in the Asian population. Instead, the G2385R mutation was reported as a major risk factor in the Asian population. Similar to other LRRK2 studies, G2385R studies have also focused on kinase activity. Here, we investigated GTPase activities of G2385R with other LRRK2 mutants, such as G2019S, R1441C, and I2020T, as well as wild type (WT). Our results suggest that both I2020T and G2385R contain GTPase activities stronger than that of WT. A kinase assay using the commercial recombinant proteins showed that I2020T harbored stronger activity, whereas G2385R had weaker activity than that of WT, as reported previously. This is the first report of LRRK2 I2020T and G2385R GTPase activities and shows that most of the LRRK2 mutations that are pathogenic or a risk factor altered either kinase or GTPase activity, suggesting that their physiological consequences are caused by altered enzyme activities.

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RIT2 reduces LRRK2 kinase activity and protects against alpha-synuclein neuropathology

10.1101/2020.10.21.348144 ◽

2020 ◽

Author(s):

Julia Obergasteiger ◽

Anne-Marie Castonguay ◽

Giulia Frapporti ◽

Evy Lobbestael ◽

Veerle Baekelandt ◽

...

Keyword(s):

Risk Factor ◽

Substantia Nigra ◽

Dopaminergic Neurons ◽

Kinase Activity ◽

Alpha Synuclein ◽

The Novel ◽

Lrrk2 Kinase ◽

Novel Strategy ◽

Lrrk2 Kinase Activity

AbstractIn Parkinson’s disease (PD) misfolded alpha-synuclein (aSyn) accumulates in the substantia nigra, where dopaminergic neurons are progressively lost. The mechanisms underlying aSyn pathology are still unclear but hypothesized to involve the autophagy lysosome pathways (ALP). LRRK2 mutations are a major cause of familial and sporadic PD, hyperactivate kinase activity and its pharmacological inhibition reduces pS129-aSyn inclusions. We observed selective downregulation of the novel PD risk factor RIT2 in G2019S-LRRK2 expressing cells. Here we studied whether RIT2 could modulate LRRK2 kinase activity. RIT2 overexpression in G2019S-LRRK2 cells rescued ALP abnormalities and diminished aSyn inclusions. In vivo, viral mediated overexpression of RIT2 operated neuroprotection against AAV-A53T-aSyn. Furthermore, RIT2 overexpression prevented the A53T-aSyn-dependent increase of LRRK2 kinase activity in vivo. Our data indicate that RIT2 inhibits overactive LRRK2 to ameliorate ALP impairment and counteract aSyn aggregation and related deficits. Targeting RIT2 could represent a novel strategy to combat neuropathology in familial and idiopathic PD.

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R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils

Acta Neuropathologica ◽

10.1007/s00401-021-02325-z ◽

2021 ◽

Author(s):

Ying Fan ◽

Raja S. Nirujogi ◽

Alicia Garrido ◽

Javier Ruiz-Martínez ◽

Alberto Bergareche-Yarza ◽

...

Keyword(s):

Peripheral Blood ◽

Kinase Activity ◽

Human Peripheral Blood ◽

Post Mortem ◽

Kinase Activation ◽

G2019s Mutation ◽

Lrrk2 G2019s ◽

Blood Neutrophils ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

AbstractHeterozygous gain-of-kinase function variants in LRRK2 (leucine-rich repeat kinase 2) cause 1–2% of all cases of Parkinson’s disease (PD) albeit with incomplete and age-dependent penetrance. All pathogenic LRRK2 mutations reside within the two catalytic domains of LRRK2—either in its kinase domain (e.g. G2019S) with modest effect or its ROC-COR GTPase domain (e.g. R1441G/H) with large effect on LRRK2 kinase activity. We have previously reported assays to interrogate LRRK2 kinase pathway activity in human bio-samples measuring phosphorylation of its endogenous substrate Rab10, that mirrors LRRK2 kinase activation status. Here, we isolated neutrophils from fresh peripheral blood from 101 participants including 42 LRRK2 mutation carriers (21 with the G2019S and 21 with the R1441G mutations), 27 patients with idiopathic PD, and 32 controls. Using a dual approach, LRRK2 dependent Rab10 phosphorylation at Threonine 73 (pRab10Thr73) was measured by quantitative multiplexed immunoblotting for pRab10Thr73/total Rab10 as well as targeted mass-spectrometry for absolute pRab10Thr73 occupancy. We found a significant over fourfold increase in pRab10Thr73 phosphorylation in carriers of the LRRK2 R1441G mutation irrespective of clinical disease status. The effect of the LRRK2 G2019S mutation did not reach statistical significance. Furthermore, we show that LRRK2 phosphorylation at Serine 935 is not a marker for LRRK2 kinase activity in human neutrophils. When analysing pRab10Thr73 phosphorylation in post-mortem brain samples, we observed overall high variability irrespective of clinical and LRRK2 mutation status and attributed this mainly to the adverse effect of the peri- and post-mortem period on the stability of posttranslational modifications such as protein phosphorylation. Overall, in vivo LRRK2 dependent pRab10Thr73 phosphorylation in human peripheral blood neutrophils is a specific, robust and promising biomarker for significant LRRK2 kinase hyperactivation, as with the LRRK2 R1441G mutation. Additional readouts and/or assays may be needed to increase sensitivity to detect modest LRRK2 kinase activation, as with the LRRK2 G2019S mutation. Our assays could be useful for patient stratification and target engagement studies for LRRK2 kinase inhibitors.

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Allosteric inhibition of LRRK2, where are we now

Biochemical Society Transactions ◽

10.1042/bst20200424 ◽

2020 ◽

Vol 48 (5) ◽

pp. 2185-2194

Author(s):

Ahmed Soliman ◽

Fatma Nihan Cankara ◽

Arjan Kortholt

Keyword(s):

Recent Progress ◽

Kinase Activity ◽

Kinase Inhibitors ◽

High Selectivity ◽

Gtpase Activity ◽

Leucine Rich Repeat ◽

Allosteric Inhibitors ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

Parkinson's disease (PD) is the second most common neurodegenerative disease. In recent years, it has been shown that leucine-rich repeat kinase 2 (LRRK2) has a crucial function in both familial and sporadic forms of PD. LRRK2 pathogenic mutations are thought to result in an increase in LRRK2 kinase activity. Thus, inhibiting LRRK2 kinase activity has become a main therapeutic target. Many compounds capable of inhibiting LRRK2 kinase activity with high selectivity and brain availability have been described. However, the safety of long-term use of these ATP-competitive LRRK2 kinase inhibitors has been challenged by several studies. Therefore, alternative ways of targeting LRRK2 activity will have a great benefit. In this review, we discuss the recent progress in the development of allosteric inhibitors of LRRK2, mainly via interfering with GTPase activity, and propose potential new intra and interprotein interactions targets that can lead to open doors toward new therapeutics.

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The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity

10.1101/2020.10.21.336578 ◽

2020 ◽

Author(s):

Adrienne E. D. Stormo ◽

Molly FitzGibbon ◽

Farbod Shavarebi ◽

Elizabeth M. Earley ◽

Lotus S. Lum ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Kinase Activity ◽

Missense Mutations ◽

Quantitative Mass Spectrometry ◽

Lrrk2 G2019s ◽

Mutant Lrrk2 ◽

Lrrk2 Kinase ◽

Regulatory Loop ◽

Familial Parkinson’S Disease ◽

Lrrk2 Kinase Activity

AbstractMissense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s Disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (Tripartite Motif Family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2822-982, a flexible interdomain region we designate the “Regulatory Loop” (RL). Phosphorylation of LRRK2 Ser910/935 within LRRK2 RL serves as a molecular switch controlling LRRK2’s association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 prevents upregulation of LRRK2 kinase activity by Rab29 and also rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, modulating its cytoskeletal recruitment, turnover, kinase activity, and cytotoxicity.

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Increased LRRK2 kinase activity alters neuronal autophagy by disrupting the axonal transport of autophagosomes

Current Biology ◽

10.1016/j.cub.2021.02.061 ◽

2021 ◽

Author(s):

C. Alexander Boecker ◽

Juliet Goldsmith ◽

Dan Dou ◽

Gregory G. Cajka ◽

Erika L.F. Holzbaur

Keyword(s):

Axonal Transport ◽

Kinase Activity ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity ◽

Neuronal Autophagy

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Vitamin B12 modulates Parkinson’s disease LRRK2 kinase activity through allosteric regulation and confers neuroprotection

Cell Research ◽

10.1038/s41422-019-0153-8 ◽

2019 ◽

Vol 29 (4) ◽

pp. 313-329 ◽

Cited By ~ 13

Author(s):

Adam Schaffner ◽

Xianting Li ◽

Yacob Gomez-Llorente ◽

Emmanouela Leandrou ◽

Anna Memou ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Vitamin B12 ◽

Kinase Activity ◽

Allosteric Regulation ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

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Ser1292 Autophosphorylation Is an Indicator of LRRK2 Kinase Activity and Contributes to the Cellular Effects of PD Mutations

Science Translational Medicine ◽

10.1126/scitranslmed.3004485 ◽

2012 ◽

Vol 4 (164) ◽

pp. 164ra161-164ra161 ◽

Cited By ~ 181

Author(s):

Z. Sheng ◽

S. Zhang ◽

D. Bustos ◽

T. Kleinheinz ◽

C. E. Le Pichon ◽

...

Keyword(s):

Kinase Activity ◽

Cellular Effects ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

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Measurement of LRRK2 Kinase Activity by Proximity Ligation Assay

BIO-PROTOCOL ◽

10.21769/bioprotoc.4140 ◽

2021 ◽

Vol 11 (17) ◽

Author(s):

Matthew Keeney ◽

Eric Hoffman ◽

J. Greenamyre ◽

Roberto Di Maio

Keyword(s):

Kinase Activity ◽

Proximity Ligation Assay ◽

Proximity Ligation ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

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Phosphorylation of LRRK2: from kinase to substrate

Biochemical Society Transactions ◽

10.1042/bst20120128 ◽

2012 ◽

Vol 40 (5) ◽

pp. 1102-1110 ◽

Cited By ~ 35

Author(s):

Evy Lobbestael ◽

Veerle Baekelandt ◽

Jean-Marc Taymans

Keyword(s):

Kinase Activity ◽

Pathological Process ◽

Rapid Decrease ◽

Leucine Rich Repeat ◽

Phosphorylated Protein ◽

Pathogenic Variants ◽

Disease Protein ◽

Lrrk2 Kinase ◽

Disease Modifying ◽

Lrrk2 Kinase Activity

The PD (Parkinson's disease) protein LRRK2 (leucine-rich repeat kinase 2) occurs in cells as a highly phosphorylated protein, with the majority of phosphosites clustering in the region between the ankyrin repeat and leucine-rich repeat domains. The observation that several pathogenic variants of LRRK2 display strongly reduced cellular phosphorylation suggests that phosphorylation of LRRK2 is involved in the PD pathological process. Furthermore, treatment of cells with inhibitors of LRRK2 kinase activity, which are currently considered as potential disease-modifying therapeutics for PD, leads to a rapid decrease in the phosphorylation levels of LRRK2. For these reasons, understanding the cellular role and regulation of LRRK2 as a kinase and as a substrate has become the focus of intense investigation. In the present review, we discuss what is currently known about the cellular phosphorylation of LRRK2 and how this relates to its function and dysfunction.

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