Name: assaying protein kinase activity with radiolabeled atp
Uploaded: 2017-05-26T14:00:00
Description: Watch this Scientific Journal Video about Assaying Protein Kinase Activity with Radiolabeled ATP at JoVE.com

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 ...

<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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protein A Sepharose CL-4B</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-0963-03</td>
		</tr>
		<tr>
			<td>Radiolabeled [&#947;-32P] ATP</td>
			<td>Perkin Elmer</td>
			<td>NEG035C010MC</td>
		</tr>
		<tr>
			<td>Laemmli Buffer</td>
			<td>Bio-Rad</td>
			<td>#1610737</td>
		</tr>
		<tr>
			<td>Radioactive shielding</td>
			<td>Research Products International</td>
			<td>BR-006</td>
		</tr>
		<tr>
			<td>R-250 dye (Coomassie)</td>
			<td>Thermo Fisher</td>
			<td>20278</td>
		</tr>
		<tr>
			<td>Whatman Grade 3 qualitative filter paper</td>
			<td>Whatman</td>
			<td>1003-917</td>
		</tr>
		<tr>
			<td>Slab gel vacuum dryer</td>
			<td>Bio-Rad</td>
			<td>Model 583 Gel Dryer #1651745&#160;</td>
		</tr>
		<tr>
			<td>Autorad marker</td>
			<td>Agilent Technologies</td>
			<td>420201</td>
		</tr>
		<tr>
			<td>Phosphorescent ruler</td>
			<td>Sigma Aldrich</td>
			<td>R8133</td>
		</tr>
		<tr>
			<td>Phosphorescent dots</td>
			<td>Sigma Aldrich</td>
			<td>L5149</td>
		</tr>
		<tr>
			<td>Geiger counter</td>
			<td>Ludlum</td>
			<td>Model 3 with 44-9 detector</td>
		</tr>
		<tr>
			<td>BioMax Cassette 8x10&#34;</td>
			<td>Carestream Health</td>
			<td>107 2263</td>
		</tr>
		<tr>
			<td>BioMax MS Film 8x10&#34;</td>
			<td>Carestream Health</td>
			<td>829 4985</td>
		</tr>
		<tr>
			<td>BioMax MS Intensifying Screen 8x10&#34;</td>
			<td>Carestream Health</td>
			<td>851 8706</td>
		</tr>
		<tr>
			<td>Medical/X-ray film processor</td>
			<td>Konica Minolta</td>
			<td>SRX-101A</td>
		</tr>
		<tr>
			<td>Scintillation vials</td>
			<td>Research Products International</td>
			<td>125500</td>
		</tr>
		<tr>
			<td>Scintillation fluid</td>
			<td>MP Biomedicals</td>
			<td>188245305</td>
		</tr>
		<tr>
			<td>Beckman LS 6500 Scintillation counter</td>
			<td>Beckman</td>
			<td>LS 6500</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 ...

The overall goal of this assay is to measure the enzymatic activity of protein kinases. This method can help answer key questions in the kinase signaling field, such as how active a kinase or kinase mutant is, its specificity, and whether its activity is sensitive to different cell treatments. The main advantage of this technique is that it is both versatile and quantitative.Demonstrating the procedure will be Steve Stippec, a research scientist at the Cobb Laboratory who has a well of experience with this assay. To begin, add two microliters of one milligrams per milliliter antibody to 200 microliters of cell lysates and incubate at four degrees Celsius for one hour while rocking. Wash protein A-Sepharose beads two to three times by adding lysis buffer and then touch spinning at four degrees Celsius for 30 seconds to one minute at 5000 times g, then remove the supernatant with a pipet and re-suspend the beads in buffer.Next, add 30 microliters of 50%slurry of protein A-Sepharose beads and lysis buffer to the lysates. Incubate the lysates at four degrees Celsius for one hour while rocking. Touch spin at four degrees Celsius to pellet the beads and remove the supernatant.Wash the beads three times with one milliliter of bead wash buffer, followed by touch spinning, then wash the beads once with one X kinase reaction buffer. After touch spinning, remove as much buffer as possible without removing the beads. To initialize the assay, add the entire reaction mixture to the kinase sample.Incubate the reaction at 30 degrees Celsius for five minutes to one hour depending on the activity of kinase being assayed. Stop the reaction by placing it on ice and adding 7.5 microliters of five X Laemmli sample buffer, then heat the reaction at 100 degrees Celsius for three to five minutes in the heat block. After touch spinning the sample, load 20 microliters per well on a 10 to 15%SDS-PAGE gel.Make sure to keep the gel apparatus shielded to limit exposure to phosphorus-32 because it is a high energy beta emitter. Run the gel long enough to separate kinase and substrate. Following the run, remove the gel from glass and aluminum plates, then place the gel in 50 milliliters of Coomassie stain for one hour on an orbital shaker set to 50 RPM.For this step, use a container that is slightly larger than the gel itself. To destain the gel, move it from the stain to the fixing solution. Place pieces of foam or knotted laboratory wipes in the container with the gel to absorb the Coomassie dye.Rock the gel in 600 to 700 milliliters of fixing solution overnight on orbital shaker at 50 RPM. The next day, remove the gel from fixing solution and soak it in 200 milliliters of methanol for one to two minutes with gentle agitation until the gel turns milky white. This will help prevent cracking during the drying step.Next, wet a roughly 14 centimeter by 14 centimeter piece of qualitative filter paper with methanol and place it onto a slab gel vacuum dryer. Lay the gel down onto the filter paper, front side facing up. Carefully cover the gel with plastic wrap, then turn on and run dryer and apply vacuum.Once the vacuum has been attained after only a few seconds, roll out air bubbles with a soft rubber brayer. Continue the run for 90 minutes. Remove the dried gel and attach an autorad marker, such as a phosphorescent ruler or dot to the filter paper on the side of the gel and place it in a cassette containing an intensifying screen.Use a Geiger counter to check the intensity of the signal. For weaker signals, exposure at minus 70 degrees Celsius to minus 80 degrees Celsius with an intensifying screen will increase the film band density. In a darkroom, place a piece of film on top of the dried gel inside the film cassette containing an intensifying screen.If the CPM count is 100 or below, try an overnight exposure at minus 70 to 80 degrees Celsius. Alternatively, if the count is closer to 10, 000, start with a one hour exposure and optimize from there. At the conclusion of the exposure, remove the film before developing it in a medical or X-ray film processor.Mark the bands corresponding to the protein standards on the film. Also, label the protein standards and the reaction lanes for future reference. Excise the bands from the gel that correspond to the bands of interest on the film.Place the bands in seven milliliter scintillation vials and add four milliliters of scintillation fluid. Count the bands with a liquid scintillation counter. Confirm that the counter is set to monitor the correct energy spectrum window for phosphorus-32.Proceed to analyze the results as described in the text protocol. Purified ERK2 fractions were used in a kinase assay with MBP in the presence or absence of MEK1R4F, a constitutively active stimulator of ERK2 kinase activity. In a similar experiment, an MBP kinase assay was used to measure activity of recombinant ERK2 mutants purified from bacterial cultures relative to wild type protein.Although ERK2 was able to phosphorylate MBP when stimulated by MEK1R4F, ERK2 kinase activity is dramatically abrogated by mutation of either the catalytic lysine or a threonine proximal to the canonical sites of dual phosphorylation. ERK2 T188D and ERK2 T188E display marginal kinase activity toward a small flexible peptide. However, they are unable to robustly phosphorylate the known ERK2 substrates, Nup153 and PDX1, as seen with wild type ERK2.Once mastered, this technique can be done in one to two days if it is performed properly. After watching this video, you should have a good understanding of how to use radiolabeled ATP to assay the activity of protein kinases to better understand the signaling networks in which they reside. While attempting this procedure, it's important to remember to optimize the amino precipitation protocol for pulling down kinases of interest to account for equal protein amounts and to measure concentrations of recombinant protein to ensure accurate comparisons of kinase activity.After its development, this technique paved the way for researchers in the field of cell signaling to explore specific transduction pathways and their roles in homeostatic regulation as well as their contributions to disease progression. Don't forget that working with radioactive materials can be extremely hazardous, and precautions, such as wearing personal protective equipment should always be taken while performing this procedure.

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