Jun 02, 2022
Assessment of PKC-dependent activation of LRRK1 in vitro
- Athanasios Karapetsas1,
- Asad Malik1,
- 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: Athanasios Karapetsas, Asad Malik, Dario Alessi 2022. Assessment of PKC-dependent activation of LRRK1 in vitro. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl89d5rv2w/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 28, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 59976
Keywords: PKC activation, lipid vesicles, Phosphorylation of LRRK1, immunoblotting analysis, ASAPCRN
Abstract
We describe a non-radioactive assay that we deploy for analysing the kinase activity of recombinant LRRK1 following in vitro activation by Protein kinase C (PKC) isoforms. This assay can also be used to analyse the effect of PKC on LRRK1 immunoprecipitated from cells.
Attachments
393-856.docx
63KB
Guidelines
Note: Once the in vitro kinase assay has been performed, we recommend analysing the reaction products by quantitative immunoblotting (as described in XXXXX).
Note: This protocol can be adapted to analyse activation of LRRK1 that has been immunoprecipitated from cells (as described in XXXXXX).
Materials
Reagents:
- Recombinant PKC isoform protein (available from MRC Reagents and Services: https://mrcppureagents.dundee.ac.uk/)
- Recombinant Rab7A protein (available from MRC Reagents and Services: https://mrcppureagents.dundee.ac.uk/)
- Recombinant LRRK1 wild type [27-2015] protein
Note
Note: Recombinant LRRK1 protein is expressed and purified by following the protocol described in: XXXXX
4. Kinase assay buffer:
| A | B | |
| HEPES pH 7.5 | 25 mM | |
| 2-mercaptoethanol | 0.1% (v/v) | |
| KCl | 50 mM | |
| CaCl2 | 1 mM | |
| MgCl2 | 10 mM | |
| ATP | 1 mM |
- L-α-Phosphatidylserine (Avanti Polar Lipids, resuspended in methanol and chloroform at a 1:1 ratio for long-term storage)
- L-α-Diacylglyerol (Avanti Polar Lipids, resuspended in methanol and chloroform at a 1:1 ratio for long-term storage)
- 4X Loading buffer: NUPAGE LDS sample buffer (4x)Thermo Fisher ScientificCatalog #NP0007 , or
4X SDS loading buffer
| A | B | |
| Tris-HCl, pH6.8 | 250mM | |
| SDS | 8% (w/v) | |
| Glycerol | 40% (v/v) | |
| Bromophenol blue | 0.02% (w/v) |
Anti-Rab7 antibody Mouse monoclonalSigma AldrichCatalog #R8779
Recombinant Anti-PKC alpha antibodyAbcamCatalog #ab11723
Equipment:
- Refrigerated bench-top centrifuge (Eppendorf microcentrifuge 5417R, or equivalent).
Equipment
Refrigerated Centrifuge
NAME
Centrifuge
TYPE
Eppendorf
BRAND
EP-5417R
SKU
LINK
- Savant SpeedVac system (Thermo #SPD140DDA, or equivalent)
- Thermo mixer (Eppendorf ThermoMixer, or equivalent)
- Disposable Glass Culture Tubes (Fisherbrand Round Bottom Disposable Borosilicate Glass Tubes, or equivalent)
Preparation of lipid vesicles for PKC activation
Preparation of lipid vesicles for PKC activation
Clean a disposable glass culture tube by washing. Allow to air-dry.
Clean a disposable glass culture tube by washing with 100% methanol. (1/3)
Clean a disposable glass culture tube by washing with 100% methanol. (2/3)
Clean a disposable glass culture tube by washing with 100% methanol. (3/3)
Pipette 0.5 µL of Diacylglycerol (stock concentration is 10 mg/mL ) and 5 µL of Phosphatidylserine (stock concentration is 10 mg/mL ) into the cleaned and dried glass tube.
Note
Note: These quantities will provide sufficient lipid vesicles for 25 reactions at a volume of 20 µL per reaction.
Vacuum dry lipids using a SpeedVac system for 00:10:00 . This should leave a visible, translucent lipid pellet.
Note
Note: Ensure that lipids are completely dried as any residual chloroform or methanol will inhibit the kinase reaction.
10m
Resuspend lipids in 50 µL of 25 millimolar (mM) HEPES 7.4 , 50 millimolar (mM) KCl. Vortex gently until pellet is no longer visible.
Kinase Reaction Step 1: Phosphorylation of LRRK1 by PKC
Kinase Reaction Step 1: Phosphorylation of LRRK1 by PKC
Note
Note: If using immunoprecipitated LRRK1 from cells, perform immunoprecipitation and washes (as described in XXXXXX) before proceeding with Step 5.
Prepare a primary “2X master mix” containing
| A | B | |
| HEPES pH 7.5 | 50 mM | |
| KCl | 100 mM | |
| 2‐Mercaptoethanol | 0.2% (v/v) | |
| MgCl2 | 20 mM | |
| ATP | 2 mM | |
| CaCl2 | 2 mM | |
| Phosphatidylserine | 200 μg/ml | |
| Diacylglycerol | 20 μg/ml |
For each reaction, add 10 µL of the primary “2X master mix” to a clean Eppendorf tube.
Add 5 µL of 200 nanomolar (nM) LRRK1 wild type protein (final concentration is 50 nanomolar (nM) ) to each reaction and allow equilibration On ice for 00:05:00 .
Note
Note: If using LRRK1 immunoprecipitated from cells, add 10 µL of the primary “2X master mix” and 5 µL of 25 millimolar (mM) HEPES 7.5 , 50 millimolar (mM) KCl, 0.1% (v/v) 2‐Mercaptoethanol to each tube containing beads-bound immunoprecipitated LRRK1.
5m
Start the kinase reaction by adding 5 µL of 400 nanomolar (nM) PKC Alpha protein (final concentration is 100 nanomolar (nM) ).
Note
Note: The final reaction volume should be 20 µL .
After 00:30:00 , transfer the Eppendorf tubes from Step 8 On ice .
30m
Kinase Reaction Step 2: Phosphorylation of Rab7A by PKC-activated LRRK1
Kinase Reaction Step 2: Phosphorylation of Rab7A by PKC-activated LRRK1
Prepare a secondary “master mix” (=Master Mix B) containing
| A | B | |
| HEPES pH 7.5 | 25 mM | |
| KCl | 50 mM | |
| MgCl2 | 10 mM | |
| ATP | 1 mM | |
| Rab7A | 1 μM |
Start the second step of the kinase reaction by adding 10 µL Master Mix B to the Eppendorf tubes from Step 5.
Transferring the Eppendorf tubes to the thermo mixer set at 30 °C , 1,000 rpm. Incubate for 00:45:00 .
45m
Stop the kinase reaction by adding 10 µL of 4× LDS (supplemented with 5% (v/v) 2‐Mercaptoethanol) loading buffer to the reaction mix to a final concentration of 1×.
If using LRRK1 immunoprecipitated from cells, stop the kinase reaction by adding 30 µL of 4× LDS loading buffer to the reaction mix to a final concentration of 2×, incubate the mixture at 70 °C on a heat block for 00:10:00 to elute LRRK1 from the resin, and collect the eluent by centrifugation through a 0.22‐μm‐pore‐size Spinex column.
10m
Incubate the samples for 00:05:00 at 70 °C on a heat block before proceeding to quantitative immunoblotting analysis.
Note
Note: If using LRRK1 immunoprecipitated from cells, supplement the samples from Step 14 with 2-Mercaptoethanol to 1% (v/v) before proceeding to Step 15.
5m
Analysis of kinase reaction products by quantitative immunoblotting analysis:
Analysis of kinase reaction products by quantitative immunoblotting analysis:
1h 15m
1h 15m
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 | |
| Antibody Target | Company | Cat. number | Host species | Dilution | |
| pS72 Rab7A | Abcam Inc. | MJF-38, Clone 1 | Rabbit | 1 mg/ml | |
| Rab7A (Total) | Sigma | R8779 | Mouse | 1.430556 | |
| LRRK1 (total) (C-terminus) | MRC-PPU Reagents and Services, University of Dundee | S405C | Sheep | 1 mg/ml | |
| PKC Alpha | Abcam Inc. | ab11723 | Mouse | 1.430556 |
Figure 1: PKC alpha dose-dependent activation of recombinant LRRK1 in vitro. Recombinant LRRK1 wild type [27-2015] was incubated with increasing concentrations of PKC Alpha (1 to 300 nM) at 30 °C for 00:30:00 with excess Mg-ATP. Reactions were subsequently incubated with 1 micromolar (µM) recombinant Rab7A and subjected to a 00:45:00 kinase reaction at 30 °C in the presence of excess Mg-ATP. Kinase reactions were subjected to immunoblot analysis with the indicated antibodies and the membranes were developed using the Odyssey CLx scan Western Blot imaging system.
1h 15m