Name: assaying the kinase activity of lrrk2 in vitro
Uploaded: 2012-01-18T13:00:00
Duration: 6 min 9 s
Description: Watch this Scientific Journal Video about Assaying the Kinase Activity of LRRK2 in vitro at JoVE.com

The author is a Parkinson's UK Research Fellow (grant F1002). This work was supported in part by the Wellcome Trust/MRC Joint Call in Neurodegeneration award (WT089698) to the UK Parkinson's Disease Consortium (UKPDC) whose members are from the UCL Institute of Neurology, the University of Sheffield and the MRC Protein Phosphorylation Unit at the University of Dundee.

Safety
The protocol described below utilizes ATP labelled with radioactive 32P at the &gamma; phosphate position to follow the kinase activity of LRRK2. It is based on the standard protocols used in our laboratory, and so with regard to many of the steps in the process such as running the gels, western blotting et cetera the materials and precise conditions should be taken as a guide as the equipment and protocols used for these processes varies from laboratory to laboratory. Compounds containing isotopes that emit ionising radiation are potentially 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 University College London and following the good laboratory practise guidelines provided by the safety services at the college (guidelines available at <a href="http://www.ucl.ac.uk/estates/safetynet/training/">http://www.ucl.ac.uk/estates/safetynet/training/</a>). Use of open source radiation should not be attempted prior to appropriate training and regulatory approval. The regulatory body responsible for open source radiation in laboratory research varies from country to country. Examples of these are: in the United Kingdom, the Health and Safety Executive (<a href="http://www.hse.gov.uk/radiation/ionising/index.htm">http://www.hse.gov.uk/radiation/ionising/index.htm</a>), in the United States the Nuclear Regulatory Commission (<a href="http://www.nrc.gov/materials/miau/regs-guides-comm.html"> http://www.nrc.gov/materials/miau/regs-guides-comm.html</a>), in Canada the Canadian Nuclear Safety Commission (<a href="http://nuclearsafety.gc.ca/eng/">http://nuclearsafety.gc.ca/eng/</a>), and in Germany Das Bundesamt f&uuml;r Strahlenschutz (<a href="http://www.bfs.de/de/bfs">http://www.bfs.de/de/bfs</a>). Users in other countries should confirm local rules, regulations and licensing authorities with their radiation safety officer. Safety precautions relevant to this protocol have been noted in the text, highlighted with the radioactive trefoil symbol (<img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" />).
1. Preparing the kinase reactions
<img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" />All reaction mixtures prepared in 1.5ml sample tubes with screw caps containing an O ring to prevent spread of radioactivity. 
<ol>
<li>Thaw protein on ice - LRRK2 wild type, D1994A, G2019S.</li>
<li>make up reaction on ice - 10nM LRRK2, 0.5&mu;g/&mu;l MBP, 5ul of 10x kinase buffer, made up to 50&mu;l with water.</li>
</ol>
2. Running the assay
<img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> All steps utilizing 32P ATP should take place in designated radiation areas.
<img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> Suitable personal protective equipment should be worn - under standard operating procedure in our laboratory these include lab coat, double gloves and protective goggles.
<img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> Samples containing 32P ATP should be shielded from users by 6mm Perspex screens to minimize exposure.
<img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> Where applicable, personal monitoring devices should be used - within UCL, any certified open source radiation user must have a film badge to monitor radiation exposure during experiments.
<img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> All experimental surfaces should be assessed for radioactive contamination before and after use using a Geiger counter.
<img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> All potentially contaminated consumables should be disposed of in strict adherence to institutional guidelines for radioactive waste disposal.
<ol>
<li>Prior to starting assay, set heating blocks to 30&deg;C and 100&deg;C respectively.</li>
<li>Remove 32P ATP from -20 freezer (note that storage conditions for 32P ATP may vary depending on supplier or type of radionucleotides used). <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> scan outside of container prior to use, thaw behind perspex screen.</li>
<li>With reactions on ice, add 1&mu;l of 32P ATP to each along with 10&mu;M of cold ATP.</li>
<li>Mix well with pipette. <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> Pulse centrifuge to bring liquid to bottom of tube, minimizing risk of contamination.</li>
<li>Remove 15&mu;l aliquot for zero time point and terminate reaction in aliquot by addition of 5&mu;l of 4x SDS sample buffer and denaturation at 100&deg;C for 10 minutes. <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> Pulse centrifuge to bring liquid to bottom of tube, minimizing risk of contamination.</li>
<li>Remaining sample placed in heating block and incubated at 30&deg;C for 60 minutes.</li>
<li>15&mu;l removed at 60 minute time point and reaction terminated by addition of 5&mu;l of 4x SDS sample buffer and denaturation at 100&deg;C for 10 minutes. <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> Pulse centrifuge to bring liquid to bottom of tube, minimizing risk of contamination.</li>
</ol>
3. Immunoblotting the samples and analysing results
<ol>
<li>Samples run on SDS-PAGE 
<ol>
 <li>10 well 4-12&#37; Bis-tris polyacrilamide gel prepared for electrophoresis using MOPS running buffer.</li>
 <li>20&mu;l of each sample loaded onto gel along with 7&mu;l of sharpstain protein standard ladder.</li>
 <li>Gel run at 160v for 90 minutes, or until dye front has reached the end of the gel. <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> All liquid in contact with radioisotopes should be treated as radioactive waste and disposed of as per institutional guidelines. UCL regulations state that liquid radioactive waste should be discarded by pouring down designated radioactive disposal sinks with copious water.</li>
</ol>
</li>
<li>Protein transferred to PVDF membrane via western blot. 
<ol>
 <li>Transfer buffer prepared, 1x Tris Glycine plus 20&#37; Methanol. PVDF Membrane and filter paper cut to correct size for gel and activated with glacial methanol in the case of the membrane or pre-wet with transfer buffer in the case of the filter paper. The membrane should be pre-labelled with ballpoint ink to allow identification of orientation. <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="Symbol 1" /> Gel removed from plastic casing and excess acrylamide removed and disposed of as radioactive waste. </li>
 <li>The gel should be formed into a sandwich with the membrane and filter paper, ensuring that no bubbles exist between the membrane and gel, and then arranged in the western blot apparatus with the membrane between the gel and the anode.</li>
 <li>Transfer carried out at 25V for 16 hours.</li>
</ol>
</li>
<li>Following protein transfer to PVDF, the membrane should be dried at room temperature. Finally, the dry membrane should be isolated between cellulose acetate sheets and exposed to either a phosphor screen or x-ray film to allow detection of radiolabelled protein. Exposure time can run from several hours to over a week depending on the specific activity of the radioisotope used and the enzyme kinetics of the reaction.</li>
<li>Phosphoscreen scanned/film processed. If using a phosphor screen, the image should be saved as a high-resolution TIFF or bitmap file, if using film then the resultant transparency should be scanned using a desktop scanner and saved as a high-resolution TIFF or bitmap file. The image can then be analyzed in ImageJ, a freeware program available on the National Institutes of Health website (<a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a>).</li>
</ol>
4. Representative Results
Figure 1 shows representative results for an assay carried out using wild type, G2019S and D1994A LRRK2 with Myelin Basic Protein as a generic phosphate acceptor substrate. Autophosphorylation of LRRK2 is visible at &asymp; 200kDa, with multiple bands representing phosphorylated MBP visible from 20-40kDa. Note the absence of autophosphorylation in the D1994A (kinase dead) lane, and increased phosphorylation due to the G2019S mutation. Note also residual phosphorylation of MBP in the kinase dead lane. This may be due to incomplete ablation of the kinase activity of LRRK2 by the D1994A mutant or reflect the presence of trace contaminating kinases in the reaction.
<img src="/files/ftp_upload/3495/3495fig1.jpg" alt="Figure 1" /> 
Figure 1.

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	<li>Lewis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+ROCO+proteins+in+health+and+disease.">The function of ROCO proteins in health and disease.</a> Biol. Cell. 101, 183-191 (2009).</li><li>Paisan-Ruiz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+the+gene+containing+mutations+that+cause+PARK8-linked+Parkinson's+disease.">Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease.</a> Neuron. 44, 595-600 (2004).</li><li>Zimprich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+LRRK2+cause+autosomal-dominant+parkinsonism+with+pleomorphic+pathology.">Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology.</a> Neuron. 44, 601-607 (2004).</li><li>West, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease-associated+mutations+in+leucine-rich+repeat+kinase+2+augment+kinase+activity.">Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity.</a> Proc. Natl. Acad. Sci. U. S. A. 102, 16842-16847 (2005).</li><li>Gloeckner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Parkinson+disease+causing+LRRK2+mutation+I2020T+is+associated+with+increased+kinase+activity.">The Parkinson disease causing LRRK2 mutation I2020T is associated with increased kinase activity.</a> Hum. Mol. Genet. 15, 223-232 (2006).</li><li>Lewis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+R1441C+mutation+of+LRRK2+disrupts+GTP+hydrolysis.">The R1441C mutation of LRRK2 disrupts GTP hydrolysis.</a> Biochem. Biophys. Res. Commun. 357, 668-671 (2007).</li><li>Greggio, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinase+activity+is+required+for+the+toxic+effects+of+mutant+LRRK2/dardarin.">Kinase activity is required for the toxic effects of mutant LRRK2/dardarin.</a> Neurobiol. Dis. 23, 329-341 (2006).</li><li>Smith, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinase+activity+of+mutant+LRRK2+mediates+neuronal+toxicity.">Kinase activity of mutant LRRK2 mediates neuronal toxicity.</a> Nat. Neurosci. 9, 1231-1233 (2006).</li><li>Deng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+selective+inhibitor+of+the+Parkinson's+disease+kinase+LRRK2.">Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2.</a> Nat. Chem. Biol. 7, 203-205 (2011).</li><li>Lee, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+leucine-rich+repeat+kinase-2+protect+against+models+of+Parkinson's+disease.">Inhibitors of leucine-rich repeat kinase-2 protect against models of Parkinson's disease.</a> Nat. Med. 16, 998-1000 (2010).</li><li>Nichols, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+specificity+and+inhibitors+of+LRRK2,+a+protein+kinase+mutated+in+Parkinson's+disease.">Substrate specificity and inhibitors of LRRK2, a protein kinase mutated in Parkinson's disease.</a> The Biochemical Journal. 424, 47-60 (2009).</li><li>Anand, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+leucine-rich+repeat+kinase+2:+enzymological+properties+and+novel+assays.">Investigation of leucine-rich repeat kinase 2: enzymological properties and novel assays.</a> FEBS. J. 276, 466-478 (2009).</li><li>Daniels, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insight+into+the+mode+of+action+of+the+LRRK2+Y1699C+pathogenic+mutant.">Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant.</a> Journal of Neurochemistry. 116, 304-315 (2011).</li><li>Jaleel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+phosphorylates+moesin+at+Thr558;+characterisation+of+how+Parkinson's+disease+mutants+affect+kinase+activity.">LRRK2 phosphorylates moesin at Thr558; characterisation of how Parkinson's disease mutants affect kinase activity.</a> Biochem. J. , (2007).</li><li>Qing, H., Wong, W., McGeer, E. G., McGeer, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lrrk2+phosphorylates+alpha+synuclein+at+serine+129:+Parkinson+disease+implications.">Lrrk2 phosphorylates alpha synuclein at serine 129: Parkinson disease implications.</a> Biochem. Biophys. Res. Commun. 387, 149-152 (2009).</li><li>Imai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+4E-BP+by+LRRK2+affects+the+maintenance+of+dopaminergic+neurons+in+Drosophila.">Phosphorylation of 4E-BP by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila.</a> Embo. J. 27, 2432-2443 (2008).</li><li>Greggio, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Parkinson+disease-associated+leucine-rich+repeat+kinase+2+(LRRK2)+is+a+dimer+that+undergoes+intramolecular+autophosphorylation.">The Parkinson disease-associated leucine-rich repeat kinase 2 (LRRK2) is a dimer that undergoes intramolecular autophosphorylation.</a> J. Biol. Chem. 283, 16906-16914 (2008).</li><li>Manning, G., Whyte, D. B., Martinez, R., Hunter, T., Sudarsanam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+kinase+complement+of+the+human+genome.">The protein kinase complement of the human genome.</a> Science. 298, 1912-1934 (2002).</li></ol>

No conflicts of interest declared.

This paper describes a basic protocol for assaying the kinase activity of LRRK2 using an in vitro system. In the interests of brevity, this has been limited to a one-hour end point template using a generic substrate, but the general protocol is applicable to a range of potential substrates and amenable to more sophisticated analyses examining the kinetics of the kinase activity of LRRK2. This highlights one of the key advantages of using an in vitro system to examine the kinase behaviour of a protein such as this: because there is complete control over the concentrations of the enzyme and substrate, it is possible to generate kinetic data and calculate Km and Vmax values for the reaction. For more detailed kinetic evaluations or evaluating the impact of putative inhibitors, a higher throughput system (such as that afforded by using the LRRKtide substrate, available from Invitrogen) is a superior alternative to the approach described here.
It is important, however, to recognise that the reductionist model system provided by in vitro kinase assays is but one tool to examine the biology of a kinase and its relationships with potential substrates. Data from assays such as this should be used in conjunction with other approaches to gain a comprehensive picture of how a kinase behaves in vitro and in a cellular context. For example, a putative substrate phosphorylated in vitro can be analysed by mass spectrometry to identify possible phosphosites that can then be manipulated by targeted mutagenesis (e.g. converting serine or threonine phosphate acceptor residues to none-phosphorylatable residues such as alanine) to examine the functional consequences of phosphorylation ex vivo.
A key consideration in interpreting data from an in vitro assay system such as this is likelihood of either type I or type II (false positive or false negative) errors. The reductionist nature of this system unfortunately disposes it to both - in the case of the former, having a purified putative substrate and purified kinase in artificially close proximity and at high concentration (compared to the cellular milieu) can result in artefactual phosphorylation events. Conversely, many kinases function as part of a complex within the context of the cell, and require co-factors for phosphorylation of a given substrate to occur. As noted above, a positive result from phosphorylation of a putative substrate using an in vitro assay system should then be tested in a cellular system to validate the result, and a negative result should be interpreted with caution.
In light of this, it is critical where possible to have both positive and negative controls to allow a comparative study of the activity of your kinase of interest. Careful selection of a positive control that has a known activity towards your protein of interest, along with a negative control that is unlikely to phosphorylate your putative substrate, is extremely valuable. One candidate control for LRRK2 is Receptor interacting kinase 3, which has a closely related primary sequence to the kinase domain of LRRK2 but has a very different overall domain structure18. This is available as recombinant protein from Invitrogen and is used as a standard control in our laboratory.
It should also be noted that the commercially available form of LRRK2 lacks the N-terminus of the protein and is tagged with Glutathione-s-transferase and this should be taken into consideration as a potential confounding factor when carrying out kinase assays with this protein, as it is not known what role the N-terminal of LRRK2 may play in the normal function of this protein. If, for example, the N-terminal portion of LRRK2 that is absent in the protein used in this protocol is critical for recruitment of a specific substrate to the kinase domain of LRRK2 then this will have a major impact on the observed phosphorylation of said substrate in the in vitro system described above.
Even with these caveats, however, the importance attached to biology of LRRK2 in Parkinson's research highlights the utility of this protocol as a valuable tool for investigating the behaviour of this protein in an in vitro setting.

<table border="1">
<tbody>
<tr>
<td>Reagent</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comment</td>
</tr>
<tr>
<td>LRRK2 wild type</td>
<td>Invitrogen</td>
<td>PV4873</td>
<td></td>
</tr>
<tr>
<td>LRRK2 G2019S</td>
<td>Invitrogen</td>
<td>PV4881</td>
<td></td>
</tr>
<tr>
<td>LRRK2 D1994A</td>
<td>Invitrogen</td>
<td>PV6051</td>
<td></td>
</tr>
<tr>
<td>Kinase buffer</td>
<td>Cell Signalling</td>
<td>9802S</td>
<td></td>
</tr>
<tr>
<td>MBP</td>
<td>Sigma</td>
<td>M1891</td>
<td></td>
</tr>
<tr>
<td>32P g ATP</td>
<td>Perkin Elmer</td>
<td>BLU002X500UC</td>
<td>500&micro;Ci, 30Ci /mMole</td>
</tr>
<tr>
<td>4x LDS sample buffer</td>
<td>Invitrogen</td>
<td>NP0007</td>
<td></td>
</tr>
<tr>
<td>2-Mercaptoethanol</td>
<td>Sigma</td>
<td>M6250</td>
<td></td>
</tr>
<tr>
<td>Protein standards</td>
<td>Invitrogen</td>
<td>LC5800</td>
<td></td>
</tr>
<tr>
<td>MOPS running buffer</td>
<td>Invitrogen</td>
<td>NP0001</td>
<td></td>
</tr>
<tr>
<td>Bis-tris acrylamide gel</td>
<td>Invitrogen</td>
<td>NP0321</td>
<td>4-12&#37; 1mm 10well</td>
</tr>
<tr>
<td>PVDF membrane</td>
<td>Millipore</td>
<td>IPVH00010</td>
<td></td>
</tr>
<tr>
<td>Transfer buffer</td>
<td>Invitrogen</td>
<td>NP0006</td>
<td></td>
</tr>
</tbody>
</table>
Table 1. Reagents
Distilled and de-ionized water was used for all dilution steps
<table border="1">
<tbody>
<tr>
<td>Equipment type</td>
<td>Company</td>
<td>Comment</td>
</tr>
<tr>
<td>Geiger counter</td>
<td>Mini Instruments</td>
<td></td>
</tr>
<tr>
<td>SDS PAGE tank</td>
<td>Invitrogen</td>
<td></td>
</tr>
<tr>
<td>Transfer tank</td>
<td>Invitrogen</td>
<td></td>
</tr>
<tr>
<td>Heat blocks (2)</td>
<td>Eppendorf</td>
<td></td>
</tr>
<tr>
<td>Phosphor screen</td>
<td>GE</td>
<td>X-ray film can be substituted</td>
</tr>
<tr>
<td>Phosphor imager</td>
<td>GE</td>
<td></td>
</tr>
<tr>
<td>Exposure cassette</td>
<td>GE</td>
<td></td>
</tr>
<tr>
<td>Centrifuge</td>
<td>Eppendorf</td>
<td></td>
</tr>
<tr>
<td>Perspex shielding</td>
<td></td>
<td></td>
</tr>
<tr>
<td>1.5ml tubes</td>
<td>VWR</td>
<td>Screw top with O ring</td>
</tr>
</tbody>
</table>
Table 2. Equipment

assaying the kinase activity of lrrk2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

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Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

assaying-the-kinase-activity-of-lrrk2-in-vitro

Research

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Biology

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons2. This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate3,4 and the chemoattractive activities of Netrin15 and Sonic Hedgehog (Shh)6 in the floor plate in the spinal cord.
Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels7. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag18. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons3,4 as well the necessity for this signal to direct the commissural trajectory9.

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.