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.
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.
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.
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 ().
1. Metabolic Labeling of Cells
2. Analyze Labeling of Proteins of Interest
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. 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).
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.
The authors have nothing to disclose.
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.
Name of the reagent | Company | Catalogue number | Comments (optional) |
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 |