The authors thank all current and former members of the Cobb laboratory for valuable work and discussions, and Dionne Ware for administrative assistance. These studies were supported by National Institutes of Health Grant R37 DK34128 and Welch Foundation Grant I1243 to M.H.C.

1. Kinase Purification Resources and General Immunoprecipitation Pipeline
NOTE: Kinases for use with this assay may be sourced from immunoprecipitates of cultured cells or by recombinant means such as affinity-tagged purification6. Below is a general protocol for flag-tagged immunoprecipitation that may have to be modified depending on the kinase of interest. Everything should be kept on ice when possible.
<ol>
	<li>Add 2 &#181;L of 1 mg/mL antibody to 200 &#181;L of cell lysates (usually ~1 mg/mL total protein concentration) and incubate at 4 &#176;C for 1 h while rocking.</li>
	<li>Wash protein A sepharose beads 2-3 times with lysis buffer (see below) by briefly spinning at 4 &#176;C for 30 s to 1 min at 5,000 x g (&#34;touch spin&#34;), removing supernatant with a pipette, and resuspending beads in buffer.
	<ol>
		<li>Prepare lysis buffer: 50 mM HEPES pH 7.7, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.2 mM sodium orthovanadate, 100 mM sodium fluoride, 50 mM &#946;-glycerophosphate, 0.1% Triton X 100 or 0.1% NP-40.</li>
	</ol>
	</li>
	<li>Add 30 &#181;L of 50% slurry of protein A sepharose beads in lysis buffer to lysates; incubate at 4 &#176;C for 1 h while rocking.</li>
	<li>Touch spin at 4 &#176;C to pellet the beads and remove the supernatant.</li>
	<li>Wash 3 times with 1 mL of bead wash buffer (1 M NaCl, 20 mM Tris pH 7.4).</li>
	<li>Wash once with 1x kinase reaction buffer (10 mM HEPES pH 8.0, 10 mM MgCl2). Remove as much buffer as possible without removing beads.</li>
</ol>
2. Initializing Kinase Reactions
NOTE: For IP kinase assays, ~15 &#181;L of unsuspended beads is a typical starting sample. For recombinant protein kinase assays, typical starting amounts range from 0.1-1.0 &#181;g in 1-10 for 25-50 &#181;L final reaction volumes. Adjust the volumes of recombinant protein samples to be equal with the buffer they are stored in prior to initializing the assay. When using high amounts of kinase, include a carrier protein such as BSA to aid in maintaining protein stability for the duration of the assay.
<ol>
	<li>Prepare the 5x kinase reaction buffer given below: 
	50 mM HEPES pH 8.0 
	50 mM MgCl2 
	50 mM Benzamidine (protease inhibitor) 
	50 mM DTT (reduction agent) 
	250 &#181;M ATP (unlabeled, or &#34;cold&#34;)</li>
	<li>Keep kinase samples on ice in 1.5 mL tubes. Prepare the following reaction mixture in separate, chilled 1.5 mL tubes: 
	21 &#181;L H2O 
	6 &#181;L 5x kinase reaction buffer 
	1 &#181;L radiolabeled (&#34;hot&#34;) ATP 
	2 &#181;L substrate (~5 mg/mL) 
	NOTE: For recombinant kinase assays, volume may be adjusted to accommodate purified protein. Calculate such that final reaction volume is 30 &#181;L. Use this recipe to prepare a master mix. Upon receipt of labeled ATP, dilute stock in H2O to a specific activity of 0.01 mCi/&#181;L prior to adding to reaction mixture.</li>
	<li>To initialize the assay, add entire reaction mixture to kinase sample and incubate reaction at 30 &#176;C for 5 min to 1 h depending on activity of kinase being assayed. 
	NOTE: When working with a kinase whose activity has not been previously assayed, perform a time course of kinase activity with 5 min intervals between time points.</li>
</ol>
3. Reaction Termination and SDS-PAGE
<ol>
	<li>Stop reaction by putting on ice and adding 7.5 &#181;L 5x Laemmli sample buffer 6.</li>
	<li>Heat at 100 &#176;C for 30 s to 2 min in heat block.</li>
	<li>Touch spin and load 20 &#181;L per well on 10-15% SDS-PAGE gel. Run gel long enough to separate kinase and substrate (typically 1 h at constant power of 5 W) Make sure to keep gel apparatus shielded to limit exposure to 32P.
	<ol>
		<li>Here, use homemade gel apparatuses, but standard commercially available mini-gels are perfectly suitable. Use the dimensions as follows: 
		Gels: 8 cm width, 5.5 cm length (resolving), 1.5 mm thickness. 
		Combs: 15 wells, 1.5 mm thickness, 2.9 mm width, 16 mm depth</li>
	</ol>
	</li>
</ol>
4. Gel Staining and Drying
Caution: For all steps be sure to reduce personal exposure to 32P by using radioactive shielding and wearing personal protective equipment. For more information on specific steps some helpful references are included.
<ol>
	<li>Remove the gel from glass/alumina plates and place in 50 mL Coomassie stain (10% glacial acetic acid, 50% methanol, 0.25% R-250 dye) for 1 h on an orbital shaker set to 50 rpm. For this step use a container that is slightly larger than the gel itself.8</li>
	<li>Move the gel from stain to fixing solution (10% glacial acetic acid, 20% methanol) in order to de-stain. Rock the gel in 600-700 mL of fixing solution overnight on orbital shaker at 50 rpm. Place pieces of foam or knotted laboratory wipes in the container with the gel to absorb the Coomassie dye 8,9.</li>
	<li>Remove gel from fixing solution and soak in 200 mL methanol for 1-2 min with gentle agitation until gel turns milky white. This will help prevent cracking during the drying step.</li>
	<li>Wet a 14 cm x 14 cm piece of qualitative filter paper with methanol and place onto slab gel vacuum dryer. Lay the gel down onto the filter paper front side facing up.</li>
	<li>Cover the gel with plastic wrap carefully to avoid wrinkles and air bubbles. Run the dryer for 1.5 h at 80 &#176;C. After the vacuum has been attained open the lid and roll out any air bubbles if needed with a soft rubber brayer.</li>
</ol>
5. Autoradiogram and Scintillation Counts
<ol>
	<li>Remove dried gel and attach an autorad marker, phosphorescent ruler or dots to the filter paper on the side of the gel. If using dots make sure they are in an asymmetric pattern. This will align the gel with the film after exposure and development.</li>
	<li>Use a Geiger counter to check the intensity of the signal. For weaker signals exposure at -70 &#176;C to -80 &#176;C with an intensifying screen will increase the film band density.</li>
	<li>In a dark room put dried gel in a film cassette with film and an intensifying screen in the following order from top to bottom: gel, film, intensifying screen.</li>
	<li>Attach the intensifying screen to the lid of the cassette and tape the gel in place to make this step easier. The intensifying screen emits light when irradiated by the beta particles from the 32P. These light emissions penetrate the film more efficiently than the beta particles.
	<ol>
		<li>Make sure the wavelength emitted from the intensifying screen corresponds to a wavelength the film is most sensitive to.</li>
		<li>Use a Geiger counter to determine how long to leave exposure for: if cpm count is ~100 or below, try overnight at -70 &#176;C. If count is ~10,000, start with a 1 h exposure and optimize from there.</li>
	</ol>
	</li>
	<li>At the conclusion of the exposure remove the film and develop in a medical/X-ray film processor. If the exposure was performed at -70 to -80 &#176;C, either allow the cassette to warm to room temperature to minimize condensation on the film or remove the film immediately before condensation forms10.</li>
	<li>Lay the film over the dried gel/filter paper and align the image of the marker/dots with the markers/dots on the dried gel. Mark on the film the bands corresponding to the protein standards. Label the protein standards and which lanes the reactions are for future reference.</li>
	<li>Excise the bands from the gel that correspond to bands of interest on the film and place them in 7 mL scintillation vials. Add 4 mL of scintillation fluid and count with liquid scintillation counter. Confirm the counter is set to monitor the correct energy spectrum window for 32P. 
	NOTE: Be sure to also excise the band corresponding to substrate from the no-kinase control lane. This will be used to calculate background radiation that will be subtracted from all sample scintillation counts.</li>
</ol>
6. Analysis of Results
NOTE: The autoradiogram provides a qualitative visualization of results. For accurate quantitation, 32P incorporation can be measured with a scintillation counter. Data are usually expressed in terms of relative activity, as shown in Figure 1B. As long as uniform conditions are maintained for all samples, relative measurements of specific activity are sufficient to compare treatments.
<ol>
	<li>When calculating absolute specific activity values, perform a rigorous optimization of all reaction conditions, including kinase, substrate, and cold ATP concentrations, in order to ensure a linear range of activity over a time course (e.g. 2 x [kinase] = 2 x specific activity). For kinases with high specific activity, perform assays with increased substrate concentration in case kinase activity is limited by amount of substrate.</li>
	<li>Calculate the specific activity of kinase of interest in units of nanomoles phosphate transferred per min per mg kinase.
	<ol>
		<li>Subtract blanks from the cpm in the counted bands.</li>
		<li>Calculate the decay of hot ATP: [(1/2)^(t/t1/2)] x 0.95 where t is the number of days since the reference date (should be provided by vendor), t1/2 is the half-life of 32P, which is 14.3 days, and 0.95 reflects the efficiency of the scintillation counter. Example: if using hot ATP that is 28 days old, the decay value should be 0.245.</li>
		<li>Calculate the picomoles (pmol) of total ATP in the assay (usually this is equal to the amount of cold ATP in the reaction): assay volume (&#181;L) x [cold ATP] (&#181;M) = total ATP (pmols). Example: 30 &#181;L x 50 &#181;M = 1500 pmol</li>
		<li>Calculate cpm/pmol ATP: [&#181;L of hot ATP x (2.2 x 107) x decay factor]/ pmol of cold ATP. 
		NOTE: The value of 2.2 x 107 converts 0.01 mCi/&#181;L of ATP to cpm.</li>
		<li>Calculate the amount of kinase in the assay in mg. For both recombinant kinase and immunoprecipitated kinases, standard methods such as BCA assays are suitable to determine starting concentrations. Example: wtERK2 0.0002 &#181;g/&#181;L in a 50 &#181;L kinase reaction is 0.01 &#181;g, or 1 x 10-5 mg.</li>
		<li>Calculate total cpm in assay. Assemble 30 &#181;L reactions and run 20 &#181;L from each reaction on a gel. Multiply the cpm counted (after blank subtraction) by a factor of total assay volume/ volume loaded. Continuing the above example, multiply all counts by 1.5, or 30/20.</li>
		<li>Calculate the specific activity of the assayed kinase: 
		Total cpm in assay)/(cpm/pmol ATP)/(assay time in minutes)/(amount of kinase in mg) 
		NOTE: Dividing the above value by 1,000 yields the specific activity in nanomoles/minute/mg</li>
	</ol>
	</li>
	<li>Calculate how many mols of phosphate are incorporated into a substrate (as long as its molecular weight is known). This is used to determine the number of phosphosites on a particular protein once the reaction has gone to completion. 
	Total cpm in assay)/(cpm/pmol ATP)]/(amount of substrate in pmols)</li>
</ol>

<ol>
	<li>Raman, M., Chen, W., Cobb, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+and+properties+of+MAPKs.">Differential regulation and properties of MAPKs.</a> Oncogene. 26 (22), 3100-3112 (2007).</li><li>Wellbrock, C., Arozarena, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Complexity+of+the+ERK/MAP-Kinase+Pathway+and+the+Treatment+of+Melanoma+Skin+Cancer.">The Complexity of the ERK/MAP-Kinase Pathway and the Treatment of Melanoma Skin Cancer.</a> Front Cell Dev Biol. 4, 33 (2016).</li><li>Yang, T., Terman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+PKA-Mediated+Phosphorylation+of+Plexin+Using+Purified+Proteins.">Characterizing PKA-Mediated Phosphorylation of Plexin Using Purified Proteins.</a> Methods Mol Biol. 1493, 147-159 (2017).</li><li>Ross, H., Armstrong, C. G., Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+method+for+the+assay+of+many+serine/threonine-specific+protein+kinases.">A non-radioactive method for the assay of many serine/threonine-specific protein kinases.</a> Biochem J. 366 (Pt 3), 977-981 (2002).</li><li>Akita, S., Umezawa, N., Kato, N., Higuchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array-based+fluorescence+assay+for+serine/threonine+kinases+using+specific+chemical+reaction.">Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction.</a> Bioorg Med Chem. 16 (16), 7788-7794 (2008).</li><li>Dalby, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Phosphorylation:+Selected+Methods+in+Enzymology.">Protein Phosphorylation: Selected Methods in Enzymology.</a> J Am Chem Soc. 121 (51), 12215 (1999).</li><li>. Autoradiography Available from: <a target="_blank" href="https://www.nationaldiagnostics.com/electrophoresis/article/autoradiography">https://www.nationaldiagnostics.com/electrophoresis/article/autoradiography</a> (2011)</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Protein Protocols Handbook. , (2009).</li><li>Xu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WNK1,+a+novel+mammalian+serine/threonine+protein+kinase+lacking+the+catalytic+lysine+in+subdomain+II.">WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.</a> J Biol Chem. 275 (22), 16795-16801 (2000).</li><li>McReynolds, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+or+Mutation+of+the+ERK2+Activation+Loop+Alters+Oligonucleotide+Binding.">Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding.</a> Biochemistry. 55 (12), 1909-1917 (2016).</li><li>Carlson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+discovery+of+ERK2+substrates+identifies+ERK-mediated+transcriptional+regulation+by+ETV3.">Large-scale discovery of ERK2 substrates identifies ERK-mediated transcriptional regulation by ETV3.</a> Sci Signal. 4 (196), rs11 (2011).</li><li>Courcelles, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+dynamics+reveal+novel+ERK1/2+MAP+kinase+substrates+with+broad+spectrum+of+functions.">Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions.</a> Mol Syst Biol. 9, 669 (2013).</li><li>Myers, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Screening+and+Hit+Validation+of+Extracellular-Related+Kinase+5+(ERK5)+Inhibitors.">High-Throughput Screening and Hit Validation of Extracellular-Related Kinase 5 (ERK5) Inhibitors.</a> ACS Comb Sci. 18 (8), 444-455 (2016).</li></ol>

The authors have nothing to disclose.

Kinases are a diverse family of proteins that have evolved extensive functionality in numerous contexts, and kinase assays have been incredibly useful in studying several signaling proteins and have greatly contributed to our current understanding of cellular communication. Notably, the same basic assay was used in characterizing two disparate kinases despite major differences in structure and activity. WNK1 kinase contains an atypical catalytic pocket where the critical lysine has shifted to a unique position and is kno...

Protein kinases are sophisticated enzymes critical for the transmission of cellular signals into appropriate responses1. Given their roles in maintaining homeostasis and the prevention or promotion of disease states2, biochemical methods for assessing kinase activity continue to be powerful tools for delineating the particulars of eukaryotic signaling3. Although strategies utilizing phospho-specific antibodies have been highly informative in terms of measuring the effects of different treatment conditions on cell signaling status4, a kinase assay allows for measuring the effects of different treatment conditions directly in terms of the enzymatic activity of a kinase of interest. While there are several options for similar assays that do not use radioactive materials5, we continue to rely on this method for robust quantitation of results. There are two typical applications of this assay, both valuable for different reasons: the immunoprecipitated (IP) kinase assay (Figure 1) and the recombinant protein kinase assay (Figure 2).
The IP kinase assay is tremendously useful for identifying factors capable of activating specific protein kinases as well as gauging inhibitory treatment conditions. Briefly, an epitope-tagged kinase of interest is transfected into cultured eukaryotic cells, subjected to a variety of treatments, immunoprecipitated, and assayed for the ability to incorporate radiolabeled phosphate into a model substrate (e.g. myelin basic protein (MBP)). IP kinase assay can also be performed without resorting to overexpression, either by immunoprecipitation of endogenous protein or any number of genome-editing techniques. Because treatments are administered in culture, this method can detect stimulations transmitted through multiple upstream factors or parallel pathways by in vitro readout. One major advantage of this method is that it does not require prior knowledge of direct upstream or downstream factors or phosphorylation sites therein. Moreover, once specific substrates for a kinase of interest have been identified, the same kinase assay protocol can be used with recombinant components to measure specific activity towards natural substrates and identify specific phosphorylation sites when combined with mass spectrometry analysis. Sites identified in this manner can be further validated with kinase assays utilizing substrate mutants. Lastly, this method can also be used to detect and measure autophosphorylation.
The protocol provided here assumes either an optimized protein purification scheme or transfection method for expressing an affinity-tagged kinase of interest in cultured cells. For more detailed expositions of transfection, lysis, immunoprecipitation, and protein purification protocols, we suggest referring to Cold Spring Harbor Protocols6. For more information regarding assay development and modification, please refer to Protein Phosphorylation: Selected Methods in Enzymology7.

<table><tbody><tr><td>Protein A Sepharose CL-4B</td><td>GE Healthcare Life Sciences</td><td>17-0963-03</td><td></td></tr><tr><td>Radiolabeled [&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P] ATP</td><td>Perkin Elmer</td><td>NEG035C010MC</td><td></td></tr><tr><td>Laemmli Buffer</td><td>Bio-Rad</td><td>#1610737</td><td></td></tr><tr><td>Radioactive shielding</td><td>Research Products International</td><td>BR-006</td><td></td></tr><tr><td>R-250 dye (Coomassie)</td><td>Thermo Fisher</td><td>20278</td><td></td></tr><tr><td>Whatman Grade 3 qualitative filter paper</td><td>Whatman</td><td>1003-917</td><td></td></tr><tr><td>Slab gel vacuum dryer</td><td>Bio-Rad</td><td>Model 583 Gel Dryer #1651745&amp;nbsp;</td><td></td></tr><tr><td>Autorad marker</td><td>Agilent Technologies</td><td>420201</td><td></td></tr><tr><td>Phosphorescent ruler</td><td>Sigma Aldrich</td><td>R8133</td><td></td></tr><tr><td>Phosphorescent dots</td><td>Sigma Aldrich</td><td>L5149</td><td></td></tr><tr><td>Geiger counter</td><td>Ludlum</td><td>Model 3 with 44-9 detector</td><td></td></tr><tr><td>BioMax Cassette 8 x 10&quot;</td><td>Carestream Health</td><td>107 2263</td><td></td></tr><tr><td>BioMax MS Film 8&quot; x 10&quot;</td><td>Carestream Health</td><td>829 4985</td><td></td></tr><tr><td>BioMax MS Intensifying Screen 8&quot; x 10&quot;</td><td>Carestream Health</td><td>851 8706</td><td></td></tr><tr><td>Medical/X-ray film processor</td><td>Konica Minolta</td><td>SRX-101A</td><td></td></tr><tr><td>Scintillation vials</td><td>Research Products International</td><td>125500</td><td></td></tr><tr><td>Scintillation fluid</td><td>MP Biomedicals</td><td>188245305</td><td></td></tr><tr><td>Beckman LS 6500 Scintillation counter</td><td>Beckman</td><td>LS 6500</td><td></td></tr></tbody></table>

assaying protein kinase activity with radiolabeled atp

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

WNK1 IP kinase assays
Myc-tagged WNK1 was transfected into HEK293 cells and immunoprecipitated with an anti-Myc antibody11. The immunoprecipitate displayed kinase activity towards the model substrate MBP as well as toward itself (Figure 1A). WNK1 mutants were then tested for kinase activity towards MBP by the same method, this time employing GST-tagged constructs (Figure 1B)...

Watch this Scientific Journal Video about Assaying Protein Kinase Activity with Radiolabeled ATP at JoVE.com

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

assaying-protein-kinase-activity-with-radiolabeled-atp

Research

Education

JoVE Core

JoVE Journal

Molecular Biology

Biochemistry

Unit 1

Protein Function

SCIENTISTS IN ACTION

18.3K Views.  University of Texas Southwestern Medical Center.  Proteïne kinasen zijn hoog geëvolueerde signaal enzymen en steigers die kritisch zijn voor inter- en intracellulaire signaaltransductie. Wij presenteren een protocol voor het meten van kinase-activiteit door middel van radioactief gemerkte adenosintrifosfaat ([γ-32P] ATP), een betrouwbare methode om te helpen bij het ophelderen van cellulaire signalering regulering. 

Video: Assaying Protein Kinase Activity with Radiolabeled ATP

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 &#176;C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 &#177; 0.10 mol/min/mol kinase and the average Km was around 0.5 USA &#181;M at 25 &#176;C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.

This study describes protocols for nonradiometric methods, a bioluminescent ADP detection assay and a phosphate-affinity SDS-PAGE, to determine the kinase activity of cardiac myosin light chain kinase (cMLCK) and the phosphorylation level of its substrate, myosin regulatory light chain (MLC2v).

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

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.

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.

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 ...

Biology

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

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

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 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, ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

Assaying Protein Kinase Activity with Radiolabeled ATP

Samenvatting

Meer video's verkennen