Sep 23, 2022
Assay for PhosphoRab activation of LRRK2 Kinase
- Claire Chiang1,
- Suzanne Pfeffer1
- 1Department of Biochemistry, Stanford University School of Medicine
- Team Alessi
Protocol Citation: Claire Chiang, Suzanne Pfeffer 2022. Assay for PhosphoRab activation of LRRK2 Kinase. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4o8zgmk/v1
Manuscript citation:
https://elifesciences.org/articles/79771
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 23, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70458
Keywords: Rab phosphorylation, LRRK2 kinase, ASAPCRN, phosphorab activation of lrrk2 kinase mst kinase, mst3 phosphorylation of rab gtpase, lrrk2 kinase mst kinase, subsequent activation of lrrk2 kinase, lrrk2 kinase, rab protein, mst3 phosphorylation, rab proteins at the same site, phosphorab activation, phosphorab8a protein, rab gtpase, contaminating mst3, use of the phosphorab, rab8a, phosphorab, lrrk2, rab10
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: 000463
Abstract
MST kinase phosphorylates Rab proteins at the same site as LRRK2 and has been used to phosphorylate Rab8A and Rab10 quantitatively. This protocol includes a method to produce phosphoRab8A protein and remove as much contaminating MST3 as possible, to enable use of the phosphoRab to test subsequent activation of LRRK2 kinase. See these references for details on MST3 phosphorylation of Rab GTPases:
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Materials
His-Rab8A Q67L full length
His-GFP-Rab10 Q68L 1-181
His-MST3 protein (pET15b 6HIS MST3 TV1; MRC-PPU DU62980)
GST-PreScission protease (plasmid MRC-PPU DU2328)
Reaction buffer: 50 mM HEPES 8 , 150 mM NaCl, 10 mM MgCl2, 0.2 mM TCEP, 250 μM GTP, 2 mM ATP, 5% (v/v)
glycerol, 5 μM BSA
LRRK2 G2019S (Thermo Fisher Scientific #A15200)
Troubleshooting
To cleave the HIS tag from MST3, bind GST-PreScission protease (50 µg ) to 100 µL glutathione agarose slurry (pre-washed and pelleted) 02:00:00 at 4 °C in a total volume of 500 µL . Wash the resin 3X with 1 mL reaction buffer. Add His-MST3 kinase (0.5 mg ) and incubate Overnight on a rotator at 4 °C .
1d
Spin beads 3000 rpm, 4°C, 00:05:00 and collect supernatant, which contains free MST3 and uncleaved His-MST3. Bind supernatant to Nickel-NTA agarose (100 µL of a 50% slurry, prewashed and pelleted) for 02:00:00 at 4 °C to trap uncleaved His-MST3.
2h 5m
Spin beads 3000 rpm, 4°C, 00:05:00 and collect supernatant, which contains free MST3 without the HIS tag.
5m
Phosphorylate His-Rab8A Q67L full length with free MST3 using a molar ratio of 1:6 (kinase:substrate) at 30 °C Overnight in reaction buffer.
Phosphorylated His-Rab8A is then separated from MST3 by gel filtration using a 24 mL Superdex 75 10/300 column (Cytiva Life Sciences, #17517401). Collect the relevant Rab-containing fractions determined by SDS-PAGE.
16h
Further purify His-Rab8A by binding to Nickel-NTA agarose (100 µL of a 50% slurry, prewashed and pelleted) and elute with 500 millimolar (mM) imidazole (3 X 100 µL ) after washing with 60 column volumes of reaction buffer.
Incubate 88 nanomolar (nM) LRRK2 G2019S with 3 micromolar (µM) His-GFP-Rab10 Q68L 1-181 or His-SUMO-Rab10 wild type full length substrate ± 6 micromolar (µM) phosphoRab8A Q67L in reaction buffer in a total volume of 100 µL .
Incubate reaction in a 30 °C water bath and collect 20 µL time points at 0, 10, and 20 minutes. Stop reactions with addition of 5 µL SDS-PAGE sample buffer. Add 200 nanomolar (nM) MLi-2 for control conditions to ensure that pRab10 detected is due to LRRK2 activity.
Analyze samples by SDS-PAGE and immunoblot for phosphoRab10; image blot with Li-COR and quantify bands using ImageJ (see below for more details).
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Citations
Axel Knebel, Kerryn Berndsen, Pawel Lis, Paul Davies, Dario R Alessi. Expression and purification of Rab8A (1-181) stoichiometrically phosphorylated at pThr72 (the LRRK2 site)
dx.doi.org/10.17504/protocols.io.butinwkeBerndsen K, Lis P, Yeshaw WM, Wawro PS, Nirujogi RS, Wightman M, Macartney T, Dorward M, Knebel A, Tonelli F, Pfeffer SR, Alessi DR. PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins.
https://doi.org/pii:e50416.10.7554/eLife.50416Dhekne HS, Yanatori I, Vides EG, Sobu Y, Diez F, Tonelli F, Pfeffer SR. LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade.
https://doi.org/pii:e202101050.10.26508/lsa.202101050Step 11
Francesca Tonelli, Dario Alessi. Quantitative Immunoblotting Analysis of LRRK2 Signalling Pathway
dx.doi.org/10.17504/protocols.io.bsgrnbv6