Detection of endogenous S1292 LRRK2 autophosphorylation in mouse tissue as a readout for kinase activity

Kluss, Jillian H.; Conti, Mary Anne; Kaganovich, Alice; Beilina, Aleksandra; Melrose, Heather L.; Cookson, Mark; Mamais, Adamantios

doi:10.1038/s41531-018-0049-1

Cited by 62 publications

(70 citation statements)

References 22 publications

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“…The LRRK2 autophosphorylation site Ser1292 has been widely used for assessing LRRK2 kinase activity [16][17][18][19] . However, its phosphorylation levels appear to be extremely low and there is no sensitive phospho-specific antibody available to reliably detect and quantify phosphorylation at this site 6,20,21 . Instead, we recently developed several highaffinity antibodies for detecting pRab proteins in cells and in tissues 14 .…”

Section: Introductionmentioning

confidence: 99%

Accurate MS-based Rab10 phosphorylation stoichiometry determination as readout for LRRK2 activity in Parkinson’s disease

Karayel

Tonelli

Winter

et al. 2019

Preprint

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Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. However, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. We developed an ultra-sensitive targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and nonphosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in human neutrophils before and after LRRK2 inhibition. Compared to healthy controls, neutrophils of LRRK2 G2019S and VPS35 D620N carriers robustly display 1.4-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.

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Section: Introductionmentioning

confidence: 99%

Accurate MS-based Rab10 phosphorylation stoichiometry determination as readout for LRRK2 activity in Parkinson’s disease

Karayel

Tonelli

Winter

et al. 2019

Preprint

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“…The activity of LRRK2 is regulated by phosphorylation at ser1292 (pSer1292) and also by 14‐3‐3 proteins, which bind only to phosphorylated LRKK2 . Previous studies have used Western blotting and immunocytochemistry for detection of phosphorylated LRRK2 . Western blotting provides restricted anatomical or cellular resolution and is paired with high levels of nonspecific and off‐target binding.…”

mentioning

confidence: 59%

“…1 Previous studies have used Western blotting and immunocytochemistry for detection of phosphorylated LRRK2. [2][3][4] Western blotting provides restricted anatomical or cellular resolution and is paired with high levels of nonspecific and off-target binding. Furthermore, as LRRK2 exists at slight levels, immunocytochemical detection of pSer1292 necessitates the usage of a high antibody concentration, leading to decreased specificity.…”

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confidence: 99%

Wild‐type LRRK2 as a new potential therapeutic target in idiopathic Parkinson's disease

Moghaddam

Aarabi

2018

Movement Disorders

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“…First, we evaluated the level of phosphorylation of LRRK2 at the S1292 residue, which is an autophosphorylation site directly related to kinase activity 39 , in all our cell lines (Fig. 5A).…”

Section: Resultsmentioning

confidence: 99%

“…2), indicating that phosphorylation at this residue is not informative on kinase activity in our hands. Next, we tested the selective LRRK2 inhibitor PF-475 for its ability to induce dephosphorylation at both S1292 and S935 residues, which are both targets of pharmacological kinase inhibition 31,39 . PF-475 (300 nM-1 μM) reduced phosphorylation at both serine residues (Fig.…”

Section: Resultsmentioning

confidence: 99%

Kinase inhibition of G2019S-LRRK2 restores autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions

Obergasteiger

Frapporti

Lamonaca

et al. 2019

Preprint

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words)The Parkinson´s disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a potent modulator of autophagy and impacts on lysosome biology and function, but unclarity exists on the precise mechanics of its role and the direction of this modulation. LRRK2 is also involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and most LRRK2 familial cases manifesting with Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to reconcile these independent pieces of information to obtain a comprehensive knowledge on LRRK2-associated pathology that could also be generalized to the idiopathic disease. Here, we report a focused investigation on the role of PD-causing G2019S-LRRK2 in the autophagy-lysosome pathway in a recombinant cell line model. Initially, we evaluated the effect of LRRK2 expression on autophagy-related transcriptome. Then, we found that G2019S-LRRK2 leads to accumulation of autophagosomes with no net effect on autophagy induction. This is linked to abnormalities in lysosome morphology and proteolytic activity that are associated with a decrease in the successful formation of autolysosomes. Despite some of these features being shared by WT-LRRK2, alpha-synuclein intracellular inclusions are specifically found in G2019S-LRRK2 cells. Pharmacological kinase inhibition is capable of rescuing defects in the autophagy-lysosome pathway and reducing the number of inclusions. Notably, this effect is prevented by upstream blockade of autophagosome-lysosome fusion events, highlighting this step of the process as critical for alpha-synuclein clearance. Mutations in the Lrrk2 gene cause late-onset, familial Parkinson´s disease (PD) with variable penetrance that is clinically indistinguishable from idiopathic PD (iPD) but with pleomorphic pathology 1 . However, the G2019S mutation is singularly the most common genetic cause of PD worldwide, with an incidence up to 40% in specific populations 2,3 , and mostly presents with alpha-synuclein (aSyn) Lewy neuropathology at autopsy 4 . The gene codes for Leucine-Rich Repeat Kinase 2 (LRRK2), a large multidomain protein with two distinct enzymatic domains (GTPase and kinase) in close vicinity to each other 5 . The PDlinked mutations reside in this enzymatic core with G2019S located in the kinase domain and reported to increase kinase activity 6 . The cellular roles impacted by LRRK2 are varied, with stronger consensus on synaptic transmission 7 , vesicle trafficking 8 and autophagy 9 . Interestingly, these pathways might converge in neuronal biology and function 10 . Several independent investigations demonstrated that LRRK2 acts at different stages of the autophagy-lysosome pathway, with some conflicting results on the direction of this modulation 9 . Indications include a kinase-dependent role of LRRK2 in the modulation of...

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Detection of endogenous S1292 LRRK2 autophosphorylation in mouse tissue as a readout for kinase activity

Cited by 62 publications

References 22 publications

Accurate MS-based Rab10 phosphorylation stoichiometry determination as readout for LRRK2 activity in Parkinson’s disease

Accurate MS-based Rab10 phosphorylation stoichiometry determination as readout for LRRK2 activity in Parkinson’s disease

Wild‐type LRRK2 as a new potential therapeutic target in idiopathic Parkinson's disease

Kinase inhibition of G2019S-LRRK2 restores autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions

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