TITLE: Assessment of in vitro kinase activity of over-expressed and endogenous LRRK2 immunoprecipitated from cells
AUTHORS: Francesca Tonelli, Alexia Kalogeropulou, Dario Alessi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>We describe a non-radioactive assay that we deploy for analysing LRRK2 protein kinase activity</span><span style = "font-style:italic;"> in vitro</span><span> using Rab proteins as a substrate. This assay can be used to measure the intrinsic activity of LRRK2 immunoprecipitated from cells as well as to assess the impact of mutations on LRRK2 activity.</span></div><br/><div class = "text-block">Protocol Overview:</div><div class = "text-block">1) Transient transfection of HEK293 cells for analysis of over-expressed LRRK2 activity.</div><div class = "text-block">2) Preparation and quantification of cell lysates (from HEK293 cells over-expressing LRRK2 or MEFs).</div><div class = "text-block"><span>3) Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity </span><span style = "font-style:italic;">in vitro</span><span style = "font-style:italic;">.</span></div><div class = "text-block"><span>4) Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity</span><span style = "font-style:italic;"> in vitro</span><span style = "font-style:italic;">.</span></div><div class = "text-block"><span>5) </span><span style = "font-style:italic;">In vitro </span><span>kinase assay using LRRK2 immunoprecipitated from cells.</span></div><div class = "text-block">6) Analysis of kinase reaction products by quantitative immunoblotting analysis.</div></div>

[STEPS]
?. [Transient transfection of HEK293 cells for analysis of over-expressed LRRK2 activity in vitro:]
Transfect HEK293 cells at around 60-70% confluency. For a  dish, add  DNA (FLAG-tagged LRRK2 or FLAG empty vector) and  of  PEI solution to  of Opti-MEMTM Reduced Serum Medium and vortex for 20/30 seconds.
10
10 µg
30 µl
1 mL
Note: We recommend including a FLAG empty vector transfection as well as a FLAG-tagged LRRK2 D2017A (kinase dead) transfection to control for specificity of LRRK2 immunoprecipitation and activity.
?. [Transient transfection of HEK293 cells for analysis of over-expressed LRRK2 activity in vitro:]
Incubate at  for  to allow the DNA/PEI complexes to form.
0 Room temperature
?. [Transient transfection of HEK293 cells for analysis of over-expressed LRRK2 activity in vitro:]
Add the transfection mix to the culture medium in each dish drop by drop using a pipette and incubate cells at  after transfection.
37 °C
?. [Transient transfection of HEK293 cells for analysis of over-expressed LRRK2 activity in vitro:]
Lyse cells  - after transfection.
?. [Preparation and quantification of cell lysates (from HEK293 cells over-expressing LRRK2 or MEFs)]
Quickly rinse cells in the tissue culture dish by carefully pouring  culture media without Foetal bovine serum (FBS) into the dish.
0 Room temperature
?. [Preparation and quantification of cell lysates (from HEK293 cells over-expressing LRRK2 or MEFs)]
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: The amount of lysis buffer to use will depend on cell type. As a guideline, use 1 ml of lysis buffer for a 10 cm dish for HEK293 cells and 750 µl of lysis buffer for a 15 cm dish for MEFs.
?. [Preparation and quantification of cell lysates (from HEK293 cells over-expressing LRRK2 or MEFs)]
Immediately transfer the cell dishes to ice.
?. [Preparation and quantification of cell lysates (from HEK293 cells over-expressing LRRK2 or MEFs)]
Scrape the cells on the dish using a cell lifter (Sigma-Aldrich CLS3008, or equivalent) to ensure all cells are detached from the dish.
?. [Preparation and quantification of cell lysates (from HEK293 cells over-expressing LRRK2 or MEFs)]
Using a pipette, transfer cell lysate to an Eppendorf tube .
on ice
?. [Preparation and quantification of cell lysates (from HEK293 cells over-expressing LRRK2 or MEFs)]
Leave samples  for  to allow for efficient lysis.
on ice
?. [Preparation and quantification of cell lysates (from HEK293 cells over-expressing LRRK2 or MEFs)]
Clarify lysates by centrifugation at  for  at .
Centrifuge: 20800 34
4 °C
?. [Preparation and quantification of cell lysates (from HEK293 cells over-expressing LRRK2 or MEFs)]
Transfer the supernatants into new Eppendorf tubes and discard the pellet. Keep the tubes .
on ice
Note: Cell lysates can be snap frozen in liquid nitrogen and stored at -80oC for future use. When assessing kinase activity of LRRK2 immunoprecipitated from cells, we do not recommend more than one freeze/thaw cycle.
?. [Preparation and quantification of cell lysates (from HEK293 cells over-expressing LRRK2 or MEFs)]
Determine the protein concentration of cell lysates by Bradford assay according to the manufacturer’s instructions, performing measurements in triplicate.
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 µg/µl.
?. [Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro]
Add  of ANTI-FLAG® M2 Affinity Gel (washed 3 times in PBS and resuspended in PBS to make a 1:1 slurry so that 20 µl of 1:1 slurry correspond to 10 µl resin) to  of cell extract.
Note: When comparing multiple FLAG-tagged variants of LRRK2, we recommend assessing the levels of LRRK2 in the lysates prior to immunoprecipitation by subjecting ~10 µg cell extract to immunoblotting, normalizing total LRRK2/Tubulin levels and adjusting how much cell lysate is to be used to immunoprecipitate LRRK2 based on this quantification, to ensure that the amount of enzyme between reactions is as close as possible.
20 µl
1 mg
Note: The immunoprecipitation conditions (amount of resin and amount of cell extract to be used) might need optimisation.
?. [Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro]
Incubate at  for , under mild agitation.
4 °C
?. [Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro]
Collect the resin by centrifugation at  for  at . Discard supernatant.
Centrifuge: 2500 34
4 °C
?. [Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro]
Resuspend resin in  of lysis buffer supplemented with  NaCl.
500 µl
?. [Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro]
Repeat step 16 and 17 twice.
?. [Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro]
Collect the resin by centrifugation at  for  at . Discard supernatant.
Centrifuge: 2500 34
4 °C
?. [Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro]
Resuspend resin in  of  Tris-HCl .
500 µl
?. [Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro]
Repeat step 16 and 20.
?. [Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro]
Collect the resin by centrifugation at  for  at . Discard supernatant.
Centrifuge: 2500 34
4 °C
?. [Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro]
Resuspend the resin in  Tris-HCl  (1:1 ratio, i.e. resuspend 10 µl resin in 10 µl Tris-HCl).
?. [Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro]
Aliquot the resin into Eppendorf tubes kept  (one Eppendorf tube per reaction,  resin each).
on ice
10 µl
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
For each reaction, couple  of anti-LRRK2 antibody (UDD3, available from MRC Reagents and Services: https://mrcppureagents.dundee.ac.uk/) to  Protein A/G Sepharose resin (washed 3 times with PBS and resuspended in PBS to make a 1:1 slurry so that 20 µl of 1:1 slurry correspond to 10 µl resin) by incubating for  at  under mild agitation. Include a control where UDD3 is replaced by pre-immune IgG.
10 µg
20 µl
4 °C
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
To get rid of any unbound antibody, wash the antibody/resin complexes: collect the resin by centrifugation at  for  at , discard supernatant and resuspend in  of PBS.
Centrifuge: 2500 34
4 °C
250 µl
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Repeat step 26 twice more.
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Add the antibody/resin mix to  of cell extract per reaction.
5 mg
Note: The immunoprecipitation conditions (amount of resin and amount of cell extract to be used) might need optimisation.
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Incubate at  for , under mild agitation.
4 °C
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Collect the resin by centrifugation at  for  at . Discard supernatant.
Centrifuge: 2500 34
4 °C
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Resuspend resin in  of lysis buffer supplemented with  NaCl.
250 µl
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Repeat step 30 and 31 twice more.
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Collect the resin by centrifugation at  for  at . Discard supernatant.
Centrifuge: 2500 34
4 °C
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Resuspend resin in  of  Tris-HCl .
250 µl
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Repeat step 33 and 34.
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Collect the resin by centrifugation at  for  at . Discard supernatant.
Centrifuge: 2500 34
4 °C
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Resuspend the resin in  Tris-HCl  (1:1 ratio).
?. [Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro]
Aliquot the resin into Eppendorf tubes kept  (one Eppendorf tube per reaction,  resin each).
on ice
20 µl
?. [In vitro kinase assay using LRRK2 immunoprecipitated from cells]
Prepare a “master mix” containing  Tris-HCl ,  MgCl2,  ATP, and recombinant Rab protein.
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 LRRK2 is in the linear range. We recommend to perform a pilot study to establish the optimal parameters to use for the kinase assay under the experimental conditions of choice. Figure 1 shows the immunoblotting data for an in vitro kinase assay using LRRK2 immunoprecipitated from increasing amounts of cell lysates.
?. [In vitro kinase assay using LRRK2 immunoprecipitated from cells]
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
Centrifuge: 1000 33
?. [In vitro kinase assay using LRRK2 immunoprecipitated from cells]
Stop the kinase reaction by adding 4X LDS loading buffer to the reaction mix to a final concentration of 2X.
?. [In vitro kinase assay using LRRK2 immunoprecipitated from cells]
Incubate the mixture at  on a heat block for  to elute LRRK2 from the resin.
70 °C
?. [In vitro kinase assay using LRRK2 immunoprecipitated from cells]
Collect the eluent by centrifugation through a -pore-size Spinex column.
0.22
?. [In vitro kinase assay using LRRK2 immunoprecipitated from cells]
Supplement the samples with 2-Mercaptoethanol to 1% (v/v).
?. [In vitro kinase assay using LRRK2 immunoprecipitated from cells]
Incubate the samples for  at  on a heat block before proceeding to quantitative immunoblotting analysis.
70 °C
?. [Analysis of kinase reaction products by quantitative immunoblotting analysis]
Once the in vitro kinase assay has been performed, we recommend analysing the reaction products by quantitative immunoblotting (as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6).Figure 2: In vitro phosphorylation of recombinant Rab10 by LRRK2 immunoprecipitated from mouse embryonic fibroblasts (MEFs). Endogenous LRRK2 was immunoprecipitated from 4 mg cell extracts derived from littermate matched wild-type and LRRK2-/- (knock-out), or littermate matched wild-type and LRRK2[G2019S] KI MEFs. The immunoprecipitates were subjected to an in vitro kinase reaction at 30°C in the presence of 1 mg recombinant Rab10 and excess Mg-ATP, with or without 1µM LRRK2 inhibitor MLi-2. The reactions were terminated after 1 hour and analyzed by immunoblotting with the indicated antibodies. The membranes were developed using the Odyssey CLx scan Western Blot imaging system.
The reaction products can be analysed by quantitative immunoblotting analysis (as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6). Table 1 lists the primary antibodies that we recommend using, which include antibodies to detect Rab phosphorylation, as well as LRRK2 auto-phosphorylation sites.