1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
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
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.
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).
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.