Jun 02, 2022
LRRK1 Immunoprecipitation kinase assay
- Asad Malik1,
- Athanasios Karapetsas1,
- Dario Alessi1
- 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
External link: https://doi.org/10.1042/BCJ20220308
Protocol Citation: Asad Malik, Athanasios Karapetsas, Dario Alessi 2022. LRRK1 Immunoprecipitation kinase assay. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3p2wpl25/v1
Manuscript citation:
Malik AU, Karapetsas A, Nirujogi RS, Chatterjee D, Phung TK, Wightman M, Gourlay R, Morrice N, Mathea S, Knapp S, Alessi DR, PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the COR GTPase domain. Biochemical Journal 479(18). doi: 10.1042/BCJ20220308
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: March 21, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 59692
Keywords: transfection of HEK293, LRRK1 activity, Immunoprecipitation, ASAPCRN
Abstract
We describe a non-radioactive assay that we deploy for analysing LRRK1 protein kinase activity in vitro using Rab7A as a substrate. This assay can be used to measure the intrinsic activity of LRRK1 immunoprecipitated from cells as well as to assess the impact of mutations on LRRK1 activity.
Attachments
382-844.docx
287KB
Guidelines
Materials
Reagents:
For transfection of GFP-tagged LRRK1 in HEK293 cells:
cDNA for expression of human GFP-tagged LRRK1 in mammalian cells; Polyethylenimine “Max” (MW 4,000) (Polysciences, Inc., cat no 24885): 1 mg/mL stock in de-ionised H2O, filtered.
Lysis buffer:
| A | B | |
| HEPES pH 7.5* | 50 mM | |
| (v/v) CHAPS hydrate* | 0.30% | |
| Na3VO4** | 1 mM | |
| NaF | 50 mM | |
| β-glycerophosphate | 10 mM | |
| sodium pyrophosphate | 5 mM | |
| sucrose | 0.27 M | |
| cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (Roche, 11836170001)** | ||
| GTP-γ-S** | 1mM | |
| Microcystin-LR (Enzo Life Sciences, ALX-350-012)** | 1 μg/ml |
*Prior optimisation has shown that LRRK1 best retains its kinase activity when cells are harvested in a lysis buffer containing 0.3% CHAPS with 50mM HEPES pH 7.5 (Figure 1).
**To be added fresh before use.
Polyethylenimine Hydrochloride Linear (MW 4000)Polysciences IncCatalog #24885
Bradford assay kit Pierce™ Coomassie Plus (Bradford) Assay KitThermo FisherCatalog #23236
Resin for LRRK1 immunoprecipitation: aGFP16-aGFP2-His6 NHS-activated Sepharose beads (available from MRC Reagents and Services: https://mrcppureagents.dundee.ac.uk/) for GFP-tagged LRRK1
IP wash buffers: Lysis buffer supplemented with 300 millimolar (mM) NaCl; 50 millimolar (mM) HEPES 7.5 .
Kinase assay buffer:
| A | B | |
| HEPES pH 7.5 | 50 mM | |
| MgCl2 | 10 mM | |
| ATP | 1 mM |
Recombinant Rab7A protein (available from MRC Reagents and Services: https://mrcppureagents.dundee.ac.uk/)
4X Loading buffer:
NUPAGE LDS sample buffer (4x)Thermo Fisher ScientificCatalog #NP0007
or
4X SDS loading buffer:
| A | B | |
| Tris-HCl, pH6.8 | 250 mM | |
| SDS | 8% (w/v) | |
| Glycerol | 40% (v/v) | |
| Bromophenol blue | 0.02% (w/v) |
Equipment:
Equipment
Refrigerated Centrifuge
NAME
Centrifuge
TYPE
Eppendorf
BRAND
EP-5417R
SKU
LINK
Plate reader for Protein quantification (BioTek Epoch, or equivalent)
Thermo mixer (Eppendorf ThermoMixer, or equivalent)
Equipment
Digital Dry Baths/Block Heaters
NAME
Dry bath/heat block
TYPE
Thermo Scientific™
BRAND
88870005
SKU
LINK
Transient transfection of HEK293 cells for analysis of over-expressed LRRK1 activity in vitro:
Transient transfection of HEK293 cells for analysis of over-expressed LRRK1 activity in vitro:
Transfect HEK293 cells at around 60-70% confluency. For a 10cm dish, add 10 µg DNA (GFP-tagged LRRK1 or GFP-empty vector) and 30 µL of 1 mg/mL PEI solution to 1 mL of Opti-MEMTM Reduced Serum Medium and vortex for 20/30 seconds.
Note
Note: We recommend including a GFP-empty vector transfection as well as a GFP-tagged LRRK1 D1409A (kinase dead) transfection to control for specificity of LRRK1 immunoprecipitation and activity.
Incubate at Room temperature for 00:20:00 to allow the DNA/PEI complexes to form.
20m
Add the transfection mix to the culture medium in each dish and incubate cells at 37 °C after transfection.
Lyse cells 20-24 hours after transfection.
Preparation and quantification of cell lysates:
Preparation and quantification of cell lysates:
Quickly rinse cells in the tissue culture dish by carefully pouring Room temperature culture media without Foetal bovine serum (FBS) into the dish.
Pour off media from the culture dish and completely aspirate any residual media. Immediately add freshly prepared ice-cold lysis buffer, ensuring that the entire surface is covered by lysis buffer.
Note
Note: The amount of lysis buffer to use will depend on cell type. As a guideline, use 400 mL of lysis buffer for a 10 cm dish for HEK293 cells.
Immediately transfer the cell dishes to ice.
Scrape the cells on the dish using a cell lifter (Sigma-Aldrich CLS3008, or equivalent) to ensure all cells are detached from the dish.
Using a pipette, transfer cell lysate to an Eppendorf tube On ice .
Leave samples On ice for 00:20:00 to allow for efficient lysis.
20m
Clarify lysates by centrifugation at 20800 x g, 4°C, 00:10:00 .
10m
Transfer the supernatants into new Eppendorf tubes and discard the pellet. Keep the tubes On ice .
Note
Note: Cell lysates can be snap frozen in liquid nitrogen and stored at -80oC for future use. When assessing kinase activity of LRRK1 immunoprecipitated from cells, we do not recommend more than one freeze/thaw cycle.
Determine the protein concentration of cell lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate.
Note
Note: Ensure the concentration of the samples is in the linear range for the Bradford assay. If it isn’t, prepare appropriate dilutions in water of each lysate. Generally, protein concentrations of near confluent cells lysed as described above should result in protein concentrations of at least 2 undetermined .
Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
Immunoprecipitation of over-expressed LRRK1 from HEK293 cells:
Note
Note: When comparing multiple GFP-tagged variants of LRRK1, we recommend assessing the levels of LRRK1 in the lysates prior to immunoprecipitation by subjecting ~10 ug cell extract to immunoblotting, normalizing total LRRK1/Tubulin levels and adjusting how much cell lysate is to be used to immunoprecipitate LRRK1 based on this quantification, to ensure that the amount of enzyme between reactions is as close as possible.
Add 20 mL of aGFP16-aGFP2-His6 NHS-activated Sepharose beads (washed 3 times in PBS and resuspended in PBS to make a 1:1 slurry) to 1 mg of cell extract.
Note
Note: The immunoprecipitation conditions (amount of resin and amount of cell extract) might need optimisation.
Incubate at 4 °C for 03:00:00 , under mild agitation.
3h
Collect the resin by centrifugation at 2500 x g, 4°C, 00:02:00 . Discard supernatant.
2m
Resuspend resin in 500 µL of lysis buffer supplemented with 300 millimolar (mM) NaCl.
Repeat steps 16 and 17 twice.
Collect the resin by centrifugation at 2500 x g, 4°C, 00:02:00 . Discard supernatant.
2m
Resuspend resin in 500 µL of 50 millimolar (mM) HEPES 7.5 .
Repeat step 16 and 20.
Collect the resin by centrifugation at 2500 x g, 4°C, 00:02:00 . Discard supernatant.
2m
Resuspend the resin in 50 millimolar (mM) HEPES 7.5 (1:1 ratio).
Aliquot the resin into Eppendorf tubes kept On ice (one Eppendorf tube per reaction, 10 µL resin each).
In vitro kinase assay:
In vitro kinase assay:
Note
The amount of kinase and that of substrate, as well as the duration of the reaction should be optimised to ensure that the measured activity of LRRK1 is in the linear range. We recommend performing a pilot study to establish the optimal parameters to use for the kinase assay under the experimental conditions of choice.
Prepare a “master mix” containing 50 millimolar (mM) HEPES 7.5 , 50 millimolar (mM) KCl, 10 millimolar (mM) MgCl2, 1 millimolar (mM) ATP, and recombinant Rab protein.
Start the kinase reaction by adding the master mix to the immunoprecipitated kinase and transferring the Eppendorf tubes to the thermo mixer set at 30 °C , 1000 rpm .
Stop the kinase reaction by adding 4X LDS loading buffer to the reaction mix to a final concentration of 2X.
Incubate the mixture at 70 °C on a heat block for 00:10:00 to elute LRRK1 from the resin.
10m
Collect the eluent by centrifugation through a 0.22‐μm‐pore‐size Spinex column.
Supplement the samples with 2‐Mercaptoethanol to 1% (v/v).
Incubate the samples for 00:05:00 at 70 °C on a heat block before proceeding to quantitative immunoblotting analysis.
5m
Analysis of kinase reaction products by quantitative immunoblotting analysis
Analysis of kinase reaction products by quantitative immunoblotting analysis
The reaction products can be analysed by quantitative immunoblotting analysis (as described in XXXX). Table 1 lists the primary antibodies that we recommend using, which include antibodies to detect Rab7A phosphorylation at Serine-72.
| A | B | C | D | E | |
| AntibodyTarget | Company | Cat. number | Host species | Dilution | |
| Rab7A (Total) | Sigma | R8779 | Mouse | 1 ug/ml | |
| alpha-tubulin | Cell Signaling Technology | 3873 | Mouse | 1:5,000 | |
| pS72 Rab7A | Abcam Inc. | MJF-38, Clone 1 | Rabbit | 1 ug/ml | |
| LRRK1 (total) (C-terminus) | MRC-PPU Reagents and Services, University of Dundee | S405C | Sheep | 1 ug/ml |