TITLE: Assessment of PKC-dependent activation of LRRK1 in vitro
AUTHORS: Athanasios Karapetsas, Asad  Malik, Dario R Alessi

[DESCRIPTION]
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.

[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).

[STEPS]
SECTION: Preparation of lipid vesicles for PKC activation
1. Clean a disposable glass culture tube by washing. Allow to air-dry.
SECTION: Preparation of lipid vesicles for PKC activation
2. 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. 
 

SECTION: Preparation of lipid vesicles for PKC activation
3. Vacuum dry lipids using a SpeedVac system for 10 min. This should leave a visible, translucent lipid pellet. 
 

SECTION: Preparation of lipid vesicles for PKC activation
4. Resuspend lipids in 50 µL of 25 millimolar (mM) HEPES pH 7.4, 50 millimolar (mM) KCl. Vortex gently until pellet is no longer visible.

SECTION: Kinase Reaction Step 1: Phosphorylation of LRRK1 by PKC
5. 
 
Prepare a primary “2X master mix” containing
 
      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  
 
SECTION: Kinase Reaction Step 1: Phosphorylation of LRRK1 by PKC
6. For each reaction, add 10 µL of the primary “2X master mix” to a clean Eppendorf tube.

SECTION: Kinase Reaction Step 1: Phosphorylation of LRRK1 by PKC
7. 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 5 min. 
 

SECTION: Kinase Reaction Step 1: Phosphorylation of LRRK1 by PKC
8. Start the kinase reaction by adding 5 µL of 400 nanomolar (nM) PKC Alpha protein (final concentration is 100 nanomolar (nM)). 
 


SECTION: Kinase Reaction Step 1: Phosphorylation of LRRK1 by PKC
9. After 30 min, transfer the Eppendorf tubes from Step 8 on ice.
SECTION: Kinase Reaction Step 2: Phosphorylation of Rab7A by PKC-activated LRRK1
10. Prepare a secondary “master mix” (=Master Mix B) containing  
 
      HEPES pH 7.5  25 mM    KCl  50 mM    MgCl2  10 mM    ATP  1 mM  
 
 
SECTION: Kinase Reaction Step 2: Phosphorylation of Rab7A by PKC-activated LRRK1
11. Start the second step of the kinase reaction by adding 10 µL Master Mix B to the Eppendorf tubes from Step 5.

SECTION: Kinase Reaction Step 2: Phosphorylation of Rab7A by PKC-activated LRRK1
12. Transferring the Eppendorf tubes to the thermo mixer set at 30 °C, 1,000 rpm. Incubate for 45 min.

SECTION: Kinase Reaction Step 2: Phosphorylation of Rab7A by PKC-activated LRRK1
13. 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×.
SECTION: Kinase Reaction Step 2: Phosphorylation of Rab7A by PKC-activated LRRK1
14. 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 10 min to elute LRRK1 from the resin, and collect the eluent by centrifugation through a 0.22‐μm‐pore‐size Spinex column.

SECTION: Kinase Reaction Step 2: Phosphorylation of Rab7A by PKC-activated LRRK1
15. Incubate the samples for 5 min at 70 °C on a heat block before proceeding to quantitative immunoblotting analysis. 
 

SECTION: Analysis of kinase reaction products by quantitative immunoblotting analysis:
16. 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. 
 
      ­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  
 
 
SECTION: Analysis of kinase reaction products by quantitative immunoblotting analysis:
17. 
 
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 30 min with excess Mg-ATP. Reactions were subsequently incubated with 1 micromolar (µM) recombinant Rab7A and subjected to a 45 min 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. 

SECTION: Preparation of lipid vesicles for PKC activation
1.1. Clean a disposable glass culture tube by washing with 100% methanol. (1/3)
SECTION: Preparation of lipid vesicles for PKC activation
1.2. Clean a disposable glass culture tube by washing with 100% methanol. (2/3)
SECTION: Preparation of lipid vesicles for PKC activation
1.3. Clean a disposable glass culture tube by washing with 100% methanol. (3/3)