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JoVE Journal Biology

Biology

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Published: September 18, 2013 doi: 10.3791/50523
Automatic Translation
1, 1, 1
1Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven and Leuven Institute for Neuroscience and Disease (LIND)

Summary

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Abstract

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Introduction

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain paralogs which share a similar domain organization. Both proteins encode a GTPase sequence akin to the Ras family of GTPases (Ras of Complex Proteins, or ROC) as well as a C-terminal of ROC domain (COR), effectively classifying both proteins to the ROCO protein family5,6. N-terminal of the ROC-COR domain tandem, both proteins encode a leucine-rich repeat domain as well as an ankyrin-like domain, while only LRRK2 encodes an extra armadillo domein6-8. C-terminal of ROC-COR, both proteins share a serine-threonine kinase domain while only LRRK2 encodes a WD40 domain in the C-terminal region8. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2 , while genetic evidence points to a role for LRRK2 in the pathogenesis of Parkinson's disease3,4 .

The phosphorylation of proteins is a common regulatory mechanism in cells. For example, phosphorylation can be essential for the activation of enzymes or for the recruitment of proteins to a signaling complex. The cellular phosphorylation of LRRK2 has been extensively characterized and phosphosite mapping has shown a majority of cellular phosphorylation sites to occur in a cluster between the ankyrin repeat and leucine rich repeat domains9-11. Although LRRK1 cellular phosphorylation sites have yet to be mapped, evidence from studies using phosphoprotein staining of blots of immunoprecipitated LRRK1 protein from COS7 cells suggests that LRRK1 protein is phosphorylated in cells12.

This paper provides a basic protocol for assaying general phosphorylation level of LRRK1 and LRRK2 in cell lines using metabolic labeling with 32P-orthophosphate. The overall strategy is straightforward. Affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. The affinity tag (3xflag) is then used to isolate the LRRK proteins from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots.

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Protocol

The present protocol uses radioactive 32P-labeled orthophosphate to follow cellular phosphorylation of LRRK2. It is important to bear in mind that all operations with radioactive reagents should be performed using appropriate protective measures to minimize exposure of radioactive radiation to the operator and the environment. Compounds containing isotopes that emit ionizing radiation can be harmful to human health and strict licensing and regulations at an institutional and national level control their use. The experiments in this protocol were carried out following training in open source radiation use at Katholieke Universiteit Leuven (KU Leuven) and following the good laboratory practice guidelines provided by the health, safety and environment department at the university. Several steps in our protocol are widely deployed such as cell culture, SDS-PAGE, western blotting and given here are details of the protocol as applied in our laboratory. It should be noted that precise experimental conditions vary from laboratory to laboratory; therefore specific measures to ensure proper handling of radioactive material should be adapted to each new laboratory setting.

Use of open source radiation is subject to prior regulatory approval and the regulatory body responsible for open source radiation in laboratory research varies from country to country. Users should consult with their institutional radiation safety officer in order to ensure that procedure conform to local rules and regulations. Information on regulatory bodies can be found: in Belgium, the Federal Agency for Nuclear Control (http://www.fanc.fgov.be, website in French or Dutch), in the United Kingdom, the Health and Safety Executive (http://www.hse.gov.uk/radiation/ionising/index.htm), in the United States the Nuclear Regulatory Commission (http://www.nrc.gov/materials/miau/regs-guides-comm.html), in Canada the Canadian Nuclear Safety Commission (http://nuclearsafety.gc.ca/eng/), and in Germany Das Bundesamt für Strahlenschutz (http://www.bfs.de/de/bfs). Safety precautions relevant to this protocol have been noted in the text, highlighted with the radioactive trefoil symbol (Rad Symbol).

1. Metabolic Labeling of Cells

  1. Prepare cells for labeling.
    1. Culture HEK293T cell lines according to standard culture conditions (37 °C, 5% CO2) in DMEM with 8% fetal calf serum and gentamycin.
    2. Expand cells sufficiently to obtain at least 1 x 106 cells per sample to test.
    3. Trypsinize cells and plate out into 6 well plates (35 mm diameter) at 106 cells/well.
    4. 24 hr after plating out cells, express 3xflag-LRRK2 protein via transfection or lentiviral vector mediated transduction.
      1. For transfection, mix per sample 4 μg DNA (pCHMWS-3xflag-LRRK2 plasmid13-15 or pCHMWS-3xflag-LRRK1 plasmid15) and 8 μl of linear polyethyleneimine (linear PEI, 1 mg/ml) into 80 μl DMEM (without additions). Allow to complex for 15-30 min then add complex to cells by mixing well into medium present.
      2. For lentiviral vector mediated transduction, dilute lentiviral vector encoding 3xflag-LRRK1 or -2 (LV-3xflag-LRRK1, LV-3xflag-LRRK2, as a rule of thumb, transduce with twice as many transducing units, i.e. number of functional vector particles, of lentivector as there are cells) into the culture medium. A description of the production of LV-3xflag-LRRK1/2 has previously been described15.
    5. When cells are 80-100% confluent (about 48 hr after transfection or transduction), rinse cells with prewarmed (37 °C) DMEM without phosphates.
  2. Label cells with 32P-ortho-phosphate.
    1. Keep in mind general principles of safety when working with radiation.
      1. Rad SymbolPerform all operations with 32P in a designated radiation area.
      2. Rad SymbolSuitable personal protective equipment should be worn - under standard operating procedure in our laboratory these include lab coat, double gloves and protective goggles.
      3. Rad SymbolAll work with 32P should be shielded from users by 6 mm Perspex screens to minimize exposure.
      4. Rad SymbolPersonal monitoring devices should always be used - within KUL all certified open source radiation user wears a film badge attached to the breast pocket of the lab coat to monitor radiation exposure during experiments.
      5. Rad Symbol All experimental surfaces should be assessed for radioactivity before and after use with a Geiger counter.
      6. Rad SymbolAll potentially contaminated consumables should be disposed of in strict adherence to institutional guidelines for radioactive waste disposal.
    2. Under a laminar flow, prepare a Falcon tube with 2.1 ml of DMEM without phosphates (prewarmed to 37 °C) per 6-well plate of cells to label.
      1. For instance, to label cells in all wells of a 6-well plate, prepare 12.6 ml medium (=6 x 2.1). This is to provide for 2 ml medium to be used per 6-well plate well of cells with a 5% excess in volume.
    3. Rad SymbolPrepare the bench at which the experiments with ionizing radiation will be performed. The working space is covered by a spill mat upon which a protective liner of absorbent material is placed. In case you are using a liner with one waterproof surface, place it with the absorbent side up.
    4. Rad SymbolAlso provide for a Perspex jar on the work space and place the tube of phosphate free medium in it.
    5. Rad SymbolTake the lead lined container with the vial of 32P labeled orthophosphate out of the fridge and bring it to the radioactivity bench. Monitor the container for external radioactive contamination using a Geiger counter.
    6. Rad SymbolDilute 32P labeled orthophosphate into the tube of DMEM without phosphates at a concentration of 24 μCi/ml.
      1. Note: at 2 ml per 6-well plate well of cells, this corresponds to 5 μCi 32P labeled orthophosphate/cm2 of cultured cells.
      2. Rad SymbolKeep the tube in the Perspex jar.
    7. Rad SymbolClose the container with the remainder of the 32P labeled orthophosphate and replace in the fridge.
    8. Rad SymbolRemove the 6-well plates with cells to be labeled from the incubator and place on the radioactivity bench.
    9. Rad SymbolRemove medium supernatant and discard. Add 2 ml of the phosphate-free medium containing 32P labeled orthophosphate/well.
    10. Rad SymbolPlace the culture plates into a Perspex box then monitor the container for external radioactive contamination using a Geiger counter.
    11. Rad SymbolTransfer the Perspex box with cells to a eukaryotic cell incubator dedicated to isotopic metabolic labeling.
    12. Rad SymbolIncubate for 1-20 hr at 37 °C in 5% CO2.
      1. In general, an incorporation time of 3 hr or more is advised. The optimal incubation time may be assessed through time course experiments for each specific protein as desired.
    13. Rad SymbolOptional: treat cells with compound.
      1. In experiments with compound treatment (such as a kinase inhibitor), a compound treatment step is included after an initial incubation time without compound to allow for the labeling. After the desired incubation time, the Perspex box containing the culture plates are removed from the incubator and brought to the radioactivity bench.
      2. Rad SymbolLabeling medium is removed and replaced by prewarmed phosphate free medium into which the compound is diluted at the desired concentration. Discard medium in a 50 ml tube for waste collection which is placed in the Perspex jar.
      3. Rad SymbolCells are replaced in the Perspex box and placed in the cell incubator for the desired contact time.
  3. Rad SymbolCollect lysates of labeled cells.
    1. Rad SymbolRemove medium from cells and discard into the waste collection tube which is placed in the Perspex jar.
    2. Rad SymbolRinse cells 2x with ice cold TBS (Tris 50 mM, NaCl 150 mM, pH 7.4, 2 ml/rinse), discarding rinse solution into a waste collection tube placed in the Perspex jar.
    3. Rad SymbolAdd 0.5 ml of ice cold immunoprecipitation (IP) lysis buffer to each well and collect lysate by pipetting lysate up and down in order to loosen all lysed cells.
      1. Prepare the required volume of IP lysis buffer (0.5 ml/sample plus 5% excess) ahead of time, adding the Protease inhibitor cocktail and phosphatase inhibitor cocktail fresh just before use.
      2. The composition of the lysis buffer is Tris 20 mM pH 7.5, NaCl 150 mM, EDTA 1 mM, Triton 1%, Glycerol 10 %, protease inhibitor cocktail and phosphatase inhibitor cocktail.
    4. Rad SymbolTransfer the lysate to a microcentrifuge tube and incubate on ice for at least 10 min.
    5. Rad SymbolCentrifuge the lysates in a microcentrifuge at >5,000 x g for 10 min.
    6. Rad SymbolDispose of radioactive waste in dedicated waste bins which are stored behind Perspex shields.

2. Analyze Labeling of Proteins of Interest

  1. Rad SymbolIsolate protein of interest by immunopurification (IP).
    1. Rad SymbolTransfer the microcentrifuge tubes with centrifuged lysates back to ice and pipette the supernatant into a microcentrifuge tube containing 10 μl bed volume of equilibrated flag-M2 agarose beads.
      1. Prepare the tubes with equilibrated flag-M2 agarose beads ahead of time.
      2. For this, pipette a volume of flag-M2 agarose slurry corresponding to 10 μl bed volume per sample plus a 5% excess.
        1. Generally, a 10 μl bed volume of beads corresponds to 20 μl slurry. Refer to the product data sheet for more details.
      3. Equilibrate the beads by rinsing 3x in 10 volumes (relative to bed volume) of IP lysis buffer.
      4. Distribute equilibrated beads evenly at 10 μl bed volume/tube into as many tubes as there are samples. Label the tubes with an identifier for each sample.
    2. Rad SymbolTransfer the microcentrifuge tubes to 50 ml tubes (about 6 microcentrifuge tubes/50 ml tube) labeled with a radioactive trefoil symbol and keep on ice.
    3. Rad SymbolTransfer samples to a rotating device behind a Perspex shield in the designated area of a cold room for end over end mixing at 4 °C for 1-20 hr.
    4. Rad SymbolTransfer the samples to a designated work space on ice.
    5. Rad SymbolSpin down the protein bound flag-M2 agarose beads in a microcentrifuge (1,000 x g, 1 min) and discard the supernatant into a waste collection tube.
    6. Rad SymbolWash the protein bound flag-M2 agarose beads by resuspending in 1 ml IP wash buffer.
      1. Composition of IP wash buffer: Tris 25 mM pH 7.5, NaCl 400 mM, Triton 1%. It is recommended to also include protease and phosphatase inhibitors in the wash buffer for proteins sensitive to degradation by co-purifying proteases or to dephosphorylation by co-purifying phosphatases.
      2. Rad SymbolSpin down the protein bound flag-M2 agarose beads in a microcentrifuge (1,000 x g, 1 min) and discard the supernatant into a waste collection tube.
      3. Rad SymbolRepeat the wash step 3x.
    7. Rad SymbolAfter the washes, resuspend the beads into 1 ml IP rinse buffer (Tris 25 mM pH 7.5, MgCl2 10 mM, dithiothreitol (DTT) 2 mM, Triton 0.02%, beta-glycerophosphate 5 mM, Na3VO4 0.1 mM).
    8. Rad SymbolSpin down the protein bound flag-M2 agarose beads in a microcentrifuge (1,000 x g, 1 min) and discard the supernatant into a waste collection tube. Remove all excess buffer.
    9. Rad SymbolResuspend beads into 40 μl of IP sample SDS loading buffer (Tris-HCl 160 mM pH 6.8, SDS 2%, DTT 0.2 M, glycerol 40%, bromophenol blue 2 mg/ml).
      1. Samples can be analyzed immediately or stored in a -20 °C freezer for ulterior analysis.
        1. Rad SymbolFor storage of samples at -20 °C, place samples in tube holders or boxes in a Perspex box in a radioactive trefoil symbol labeled freezer dedicated for storage of radioactive samples.
    10. Rad SymbolDispose of radioactive waste in dedicated waste bins which are stored behind Perspex shields.
  2. Rad SymbolResolve IP samples via SDS-PAGE and blot to PVDF membrane.
    1. Rad SymbolHeat samples in loading buffer to 95 °C for 2 min and centrifuge for 1 min at >1,000 x g to pellet the beads.
    2. Rad SymbolPrepare the protein gel electrophoresis module on the radioactivity bench behind a Perspex screen.
    3. Rad SymbolLoad samples onto a 3-8% tris-acetate SDS-PAGE gels.
      1. This type of gel is suited for resolving high molecular weight (HMW) proteins. Other gel types may also be suited, such as a 4-20% Bis-Tricine gel or Tris-glycine 4-20% gels.
      2. Include a molecular weight marker which is suitable to discern sizes of HMW proteins.
    4. Rad SymbolPerform electrophoresis at 150 V for 1 hr.
    5. Rad SymbolAfter electrophoresis, remove the gel from its plastic casing and transfer the gel to a container with Western blotting transfer buffer.
      1. Composition of Western blotting transfer buffer: Tris 50 mM, Glycine 40 mM, SDS 0.04%, Methanol 20%.
      2. Rad SymbolCut off the parts of the gel which stick out such as the well separators and bottom portion of the gel which sticks out.
    6. Prepare one polyvinylidene fluoride (PVDF) membrane per gel by dipping in methanol for 1 min, then place in transfer buffer.
      1. Membranes are cut to the same size as the gel plus a margin of 3 mm.
    7. Place a semi-dry blotting module on the radioactivity bench and remove the cover and upper electrode plate.
    8. Rad SymbolPrepare the blotting sandwich on the surface of the semi-dry blotting module.
      1. Wet an extra thick (2.5 mm thick, 7.5 x 10 cm large) blotting filter in transfer buffer and place on bottom plate of the blotting module.
      2. Rad SymbolPlace the pre-wet PVDF membrane on the blotting filter.
      3. Rad SymbolCarefully place the gel on the PVDF membrane and remove any air bubbles.
      4. Rad SymbolComplete the blot sandwich by wetting an extra thick blotting filter in transfer buffer and place on the bottom plate of the blotting module. Remove all air bubbles eventually present in the blotting sandwich.
        Please note that the description given here is compatible with the BioRad trans-blot SD system where electrodes are such that proteins migrate downwards onto the membrane. Other blotting systems are also compatible with these steps with minor adaptations such as those eventually needed to take into account another blotting direction or, in the case of tank blotting, extra liquid waste to be disposed of in the same way as the electrophoresis buffer above.
    9. Rad SymbolRemove all excess buffer with an absorbent tissue and place the top plate and the cover of the semi-dry blotting module.
    10. Rad SymbolTransfer proteins at 15 V for 1-2 hr.
    11. Rad SymbolDuring this time, clean up the electrophoresis module.
      1. Rad SymbolDispose of radioactive waste in dedicated waste bins which are stored behind Perspex shields.
      2. Rad SymbolRinse the electrophoresis module with distilled water (AD) and discard the rinse water in the radioactive liquid waste container.
    12. Rad SymbolAfter transfer, remove the PVDF membrane with blotted proteins from the blotting module.
    13. Rad SymbolOptional: perform a Ponceau S staining of blotted proteins to visualize proteins.
      1. Rad SymbolTransfer the blot to a shallow blot incubation vessel containing Ponceau S solution and incubate for 5 min.
      2. Rad SymbolRinse 2x quickly in AD.
    14. Rad SymbolDry the membrane.
  3. Rad SymbolPerform autoradiography.
    1. Rad SymbolExpose the membrane to a phosphorescence plate for 1-5 days.
    2. Rad SymbolRead the 32P off of the exposed phosphorescence plate using a Storm 840 phosphorescence scanner or equivalent and save the image as a high resolution tiff.
  4. Rad Symbol Detect protein levels via immunodetection.
    1. Rad SymbolRehydrate the membranes by dipping them briefly into methanol, then transfer to a shallow blot incubation vessel with PBS.
    2. Rad SymbolBlock the membranes in PBS-T (PBS with 0.1% Triton) containing 5% milk.
    3. Rad SymbolIncubate the blots with anti-LRRK2 antibody13,16 or anti flag antibody and process further with appropriate wash steps and secondary antibody incubation.
    4. Rad SymbolPerform chemiluminescence detection to confirm the relative protein levels of LRRK2.
  5. Quantify incorporation of 32P in LRRK2.
    1. Perform densitometric analysis of the bands on the blot autoradiograms and immunoreactivity using appropriate software such as ImageJ software, a freeware program available on the National Institutes of Health website (http://rsbweb.nih.gov/ij/).
    2. Calculate levels of phosphate incorporation as the ratio of the autoradiographic signal over the immunoreactivity level.

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Representative Results

In order to compare overall phosphorylation levels of LRRK1 and LRRK2 in cells, 3xflag tagged LRRK1 and LRRK2 were expressed in HEK293T cells15. Cells were cultured in 6-well plates and labeled with 32P and analyzed as described above in the protocol text. Figure 1 shows representative results for metabolic labeling of LRRK1 and LRRK2 in HEK293T cells. Radioactive phosphate incorporation is observed for both LRRK1 and LRRK2. Upon quantification of the 32P levels normalized to the protein levels as measured by densitometric analysis of the immunodetection with anti-flag antibody, it was found that LRRK1 had an average phosphorylation level which is lower than LRRK2 under the conditions tested, although statistical significance is not reached (P>0.05).

Figure 1
Figure 1. Metabolic labeling of LRRK1 and LRRK2. A. LRRK1 and LRRK2 expressed in HEK293T cells were metabolically labeled with 32P as described in the protocol and results sections. Depicted here are representative autoradiograms (upper panel) of the 32P incorporation as well as representative Western blots (lower panel) of LRRK1 and LRRK2 detection via their 3xflag tags. B. Quantification of the comparative metabolic labeling of LRRK1 and LRRK2 (N=4).

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Discussion

This paper provides a basic protocol for assaying general phosphorylation level of LRRK1 and LRRK2 in cell lines using metabolic labeling with 32P-orthophosphate. The overall strategy is straightforward. Affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. The affinity tag (3xflag) is then used to isolate the LRRK proteins from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and Western detection (protein signal) of the proteins on the blots. This protocol is to be distinguished from the protocol to measure LRRK2 autophosphorylation17 in that the labeling of LRRK1 or LRRK2 is performed in cell culture rather than in an in vitro phosphorylation reaction with purified proteins.

It should be noted that the detailed protocol presented here can be adjusted to accommodate for multiple variations depending on experimental needs. For instance, as labeling is efficient in most common laboratory cell lines, this protocol is not restricted to the use of the HEK293T cell line. Also, other affinity tags may be used as an alternative to 3xflag, such as HA, myc, V5, GFP or other tags18 as multiple tags can be used to efficiently immunoprecipitate LRRK1 or LRRK2. In case a protein-specific antibody is available for the protein that is suited for immunoprecipitation, as is the case for LRRK211, this can be implemented as well. With an immunoprecipitation grade protein-specific antibody, it is also feasible to perform metabolic labeling of LRRK proteins endogenously expressed in cell lines. In the case of LRRK2, several monoclonal antibodies have been described which can immunoprecipitation of endogenous LRRK219. Finally, the metabolic labeling protocol, described here for LRRK1 and LRRK2 can also be adapted to any other protein which can be immunoprecipitated from cell lines using the general strategy described above.

A key consideration before performing metabolic labeling of proteins in cell culture is how this technique compares to other methods available to determine cellular protein phosphorylation. For instance, phosphorylation at specific sites can be monitored by immunoblotting using a phospho-specific antibody. This method follows similar steps to those described here, excluding the isotopic labeling steps, and for this reason, this technique is often favored over metabolic labeling with 32P-orthophosphate when it is available. Metabolic labeling with 32P-orthophosphate provides a signal which is representative of the overall phosphorylation state of the protein, therefore it cannot provide information on the phosphorylation of specific sites. For proteins with multiple phosphorylation sites, as it is the case for LRRK210,11, the metabolic labeling technique provides a one-step assessment of the overall phosphorylation level which can ascertained with phospho-specific antibodies only pending multiple immunodetection steps. For instance, LRRK2 is highly phosphorylated in its ANK-LRR interdomain region, i.e. the S910/S935/S955/S97311,20 sites as well as in other regions10 including the recently characterized S1292 site21. In order to dissect out the roles of individual phosphosites, it is recommended to prefer experiments with phosphosite specific antibodies. For example phosphosite specific antibodies have allowed to discern that the S910/S935/S955/S973 phosphosites are dephosphorylated in several pathogenic mutants such as R1441C/G, Y1699C, I2020T, but not in the G2019S11,22, while LRRK2 disease mutant forms generally show higher phospho-S1292 levels21. However, metabolic labeling are useful for a number of other studies of cellular phosphorylation. Metabolic labeling is always an applicable technique for instance in cases of unknown phosphorylation sites, or when phospho-antibodies are not available or of low sensitivity. Finally, metabolic labeling allows comparing overall phosphorylation levels of different proteins (as shown here comparing cellular phosphorylation levels of LRRK1 and LRRK2, figure 1), a comparison which is challenging to do with phosphosite-specific antibodies given differences in sensitivity from one antibody to another.

In conclusion, the present protocol allows efficient assessment of the overall phosphorylation levels of LRRK proteins in cells. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation. Use of this protocol is recommended when phosphosite-specific antibodies are not available for the protein in study or as a step in their validation. This protocol is especially useful when the experimental goal is to compare overall phosphorylation of 2 or more different proteins as such comparisons via metabolic labeling are not biased by differences in sensitivity of detection of phosphorylation from one protein to another. Specifically for LRRK1 and LRRK2, this technique can be used to comparatively monitor activity dependent changes in phosphorylation of LRRK1 and LRRK2, given that such changes have begun to be described for LRRK211,13,23, while LRRK1 phosphorylation regulation is poorly understood.

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Disclosures

Authors have nothing to disclose.

Acknowledgments

We are also grateful to the Michael J. Fox Foundation supporting this study. We thank the Research Foundation - Flanders FWO (FWO project G.0666.09, senior researcher fellowship to JMT), the IWT SBO/80020 project Neuro-TARGET, the KU Leuven (OT/08/052A and IOF-KP/07/ 001) for their support. This research was also supported in part by the Fund Druwé-Eerdekens managed by the King Baudouin Foundation to JMT.

Materials

Name Company Catalog Number Comments
Phosphorus-32 Radionuclide, 1 mCi, buffer disodiumphosphate in 1 ml water Perkin Elmer NEX011001MC
Dulbecco’s Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) Invitrogen 11971-025 This medium contains no phosphates
Anti Flag M2 affiinty gel Sigma A2220 For an equivalent product with red colored gel (useful to more easily visualize the beads), use cat. No. F2426.
Extra thick blotting filter Bio-Rad 1703965
Ponceau S solution Sigma P7170

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References

  1. Hanafusa, H. Leucine-rich repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor. Nat Commun. 2, 158 (2011).
  2. Titz, B., et al. The proximal signaling network of the BCR-ABL1 oncogene shows a modular organization. Oncogene. 29, 5895-5910 (2010).
  3. Cookson, M. R. The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson's disease. Nat Rev Neurosci. 11, 791-797 (2010).
  4. Taymans, J. M., Cookson, M. Mechanisms of dominant parkinsonism; the toxic triangle of LRRK2, alpha-synuclein and tau. Bioessays. 32, 227-235 (2010).
  5. Lewis, P. A. The function of ROCO proteins in health and disease. Biol Cell. 101, 183-191 (2009).
  6. Marin, I., van Egmond, W. N., van Haastert, P. J. The Roco protein family: a functional perspective. FASEB J. 22, 3103-3110 (2008).
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  8. Marin, I. Ancient origin of the Parkinson disease gene LRRK2. J Mol Evol. 67, 41-50 (2008).
  9. Lobbestael, E., Baekelandt, V., Taymans, J. M. Phosphorylation of LRRK2: from kinase to substrate. Biochem Soc Trans. 40, 1102-1110 (2012).
  10. Gloeckner, C. J., et al. Phosphopeptide Analysis Reveals Two Discrete Clusters of Phosphorylation in the N-Terminus and the Roc Domain of the Parkinson-Disease Associated Protein Kinase LRRK2. J Proteome Res. 9, 1738-1745 (2010).
  11. Nichols, R. J., et al. 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization. Biochem J. 430, 393-404 (2010).
  12. Greggio, E., et al. Mutations in LRRK2/dardarin associated with Parkinson disease are more toxic than equivalent mutations in the homologous kinase LRRK1. J.Neurochem. 102, 93-102 (2007).
  13. Taymans, J. M. LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding. PLoS One. 6, 23207-23 (2011).
  14. Daniëls, V. Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant. J Neurochem. 116, 304-315 (2011).
  15. Civiero, L. Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers. PLoS One. 7, e43472 (2012).
  16. Taymans, J. M., Van den Haute, C., Baekelandt, V. Distribution of PINK1 and LRRK2 in rat and mouse brain. J.Neurochem. 98, 951-961 (2006).
  17. Lewis, P. A. Assaying the kinase activity of LRRK2 in vitro. J Vis Exp. , (2012).
  18. Lobbestael, E. Immunohistochemical detection of transgene expression in the brain using small epitope tags. BMC Biotechnol. 10, 16 (2010).
  19. Davies, P., et al. Comprehensive Characterization and Optimization of Leucine Rich Repeat Kinase 2 (LRRK2) Monoclonal Antibodies. Biochem J. , (2013).
  20. West, A. B. Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity. Hum.Mol.Genet. 16, 223-232 (2007).
  21. Sheng, Z., et al. Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations. Sci Transl Med. 4, 164ra161 (2012).
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  23. Dzamko, N., et al. Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J. 430 (910), 405-413 (2010).

Tags

Metabolic Labeling Leucine Rich Repeat Kinases LRRK1 LRRK2 Radioactive Phosphate Serine-threonine Kinase Domain Ras Of Complex Proteins Domain C-terminal Of ROC Domain Tyrosine Kinase Receptor Signaling Parkinson's Disease Phosphorylation Levels Affinity Tagged LRRK Proteins HEK293T Cells 32P-orthophosphate Assimilation Immunoprecipitation SDS-PAGE PVDF Membranes Autoradiography Western Detection
Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate
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Taymans, J. M., Gao, F., Baekelandt, … More

Taymans, J. M., Gao, F., Baekelandt, V. Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate. J. Vis. Exp. (79), e50523, doi:10.3791/50523 (2013).

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