Sep 23, 2022

Public workspaceAssay for PhosphoRab activation of LRRK2 Kinase

DOI
dx.doi.org/10.17504/protocols.io.6qpvr4o8zgmk/v1
  • 1Department of Biochemistry, Stanford University School of Medicine
Suzanne R Pfeffer
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr4o8zgmk/v1
Protocol Citation: Claire Y Chiang, Suzanne R 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
Funders Acknowledgement:
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:
CITATION
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).
LINK
dx.doi.org/10.17504/protocols.io.butinwke

CITATION
Berndsen K, Lis P, Yeshaw WM, Wawro PS, Nirujogi RS, Wightman M, Macartney T, Dorward M, Knebel A, Tonelli F, Pfeffer SR, Alessi DR (2019). PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins.. eLife.
LINK
https://doi.org/pii:e50416.10.7554/eLife.50416

CITATION
Dhekne HS, Yanatori I, Vides EG, Sobu Y, Diez F, Tonelli F, Pfeffer SR (2021). LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade.. Life science alliance.
LINK
https://doi.org/pii:e202101050.10.26508/lsa.202101050

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 Ph8 , 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)
To cleave the HIS tag from MST3, bind GST-PreScission protease (Amount50 µg ) to Amount100 µL glutathione agarose slurry (pre-washed and pelleted) Duration02:00:00 at Temperature4 °C in a total volume of Amount500 µL . Wash the resin 3X with Amount1 mL reaction buffer. Add His-MST3 kinase (Amount0.5 mg ) and incubate DurationOvernight on a rotator at Temperature4 °C .

1d
Spin beads Centrifigation3000 rpm, 4°C, 00:05:00 and collect supernatant, which contains free MST3 and uncleaved His-MST3. Bind supernatant to Nickel-NTA agarose (Amount100 µL of a 50% slurry, prewashed and pelleted) for Duration02:00:00 at Temperature4 °C to trap uncleaved His-MST3.

2h 5m
Spin beads Centrifigation3000 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 Temperature30 °C DurationOvernight in reaction buffer.

Phosphorylated His-Rab8A is then separated from MST3 by gel filtration using a Amount24 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 (Amount100 µL of a 50% slurry, prewashed and pelleted) and elute with Concentration500 millimolar (mM) imidazole (3 X Amount100 µL ) after washing with 60 column volumes of reaction buffer.

Incubate Concentration88 nanomolar (nM) LRRK2 G2019S with Concentration3 micromolar (µM) His-GFP-Rab10 Q68L 1-181 or His-SUMO-Rab10 wild type full length substrate ± Concentration6 micromolar (µM) phosphoRab8A Q67L in reaction buffer in a total volume of Amount100 µL .

Incubate reaction in a Temperature30 °C water bath and collect Amount20 µL time points at 0, 10, and 20 minutes. Stop reactions with addition of Amount5 µL SDS-PAGE sample buffer. Add Concentration200 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).
CITATION
Francesca Tonelli, Dario Alessi. Quantitative Immunoblotting Analysis of LRRK2 Signalling Pathway.
LINK
dx.doi.org/10.17504/protocols.io.bsgrnbv6

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.butinwke
Berndsen 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.50416
Dhekne 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.202101050
Step 11
Francesca Tonelli, Dario Alessi. Quantitative Immunoblotting Analysis of LRRK2 Signalling Pathway
dx.doi.org/10.17504/protocols.io.bsgrnbv6