This work was supported by NSERC grant 386634. We would like to thank members of the Screaton Lab for helpful discussions.

1. Preparation of Reagents, Plates, and Cells
<ol>
	<li>Make 500 ml lysis buffer: 25 mM Tris pH 7.5, 150 mM NaCl, 50 mM NaF, 0.5 mM EDTA pH 8.0, 0.5% TritonX-100, 5 mM beta-glycerophosphate, 5% glycerol. Store at 4 &#176;C. Immediately prior to use, add 1 mM dithiothreitol (DTT), 1 mM phenylmethyl sulfonyl fluoride (PMSF), and 1 mM sodium vanadate. After this step, PMSF is not required in any rinse buffer.</li>
	<li>Make 20 ml of 10x Kinase Buffer: 200 mM Tris pH 7.5, 50 mM beta-glycerophosphate (FW 216), 2 mM sodium vanadate. Aliquot in 1 ml tubes and store at -20 &#176;C. Immediately prior to use, add 5 mM DTT.</li>
	<li>Make 20 ml of 10x M-ATP buffer: 300 &#181;M adenosine triphosphate (ATP), 66 mM MgCl2, 33 mM MnCl2. Aliquot in 1 ml tubes and store at -20 &#176;C.</li>
	<li>Make 100 ml of 2x sodium dodecyl sulfate (SDS) lysis buffer: 1.5 g TRIS base, 20 ml glycerol, 30 ml H2O. Dissolve and adjust pH to 6.8 with HCl. Add 40 ml 10% SDS, adjust volume to 100 ml. Add 25 mg bromophenol blue.</li>
	<li>Spot 4 &#181;l of 25 ng/&#181;l mammalian expression plasmid encoding a GST-kinase 14 per well in sets of 96 well plates and label plates accordingly. Seal and freeze plates at -20 &#176;C until use.</li>
	<li>1-2 days prior to transfection, culture HEK293T cells in complete Dulbecco&#8217;s modified Eagle medium (DMEM) + 10% fetal bovine serum (FBS) and antibiotics in a 37 &#176;C incubator supplemented with CO2 (final 5%). Passage with trypsin and do not allow the stock to become &#62;80% confluent during expansion. A minimum of 6 x 107 cells are required for the screen (approximately 3 x 15 cm dishes of HEK293T cells at 80% confluence).</li>
</ol>
2. Transfection
Note: See Figure 1 for a flowchart of entire protocol.
<ol>
	<li>If plates containing kinase plasmids have been frozen, thaw at room temperature and centrifuge at 1,900 x g for 3 min to collect any moisture at the bottom of wells.</li>
	<li>Mix 8.6 ml of reduced serum medium (e.g., OPTI-MEM) with 312.7 &#181;l of lipid-based transfection reagent. Let sit for 5 min.</li>
	<li>Add 10 &#181;l of reduced serum medium to each well using an automated liquid dispenser fitted with a small volume cassette.</li>
	<li>Add 10 &#181;l per well of the reduced serum medium/transfection reagent mix from step 2.2 using an automated liquid dispenser fitted with a small volume cassette. Let sit for 20 to 45 min.</li>
	<li>Resuspend 293T cells at 7.5 x 105 cells/ml in 80 ml of complete DMEM. Add 100 &#181;l of cell suspension (i.e. 7.5 x 104 cells) per well using an automated liquid dispenser fitted with a standard volume cassette.</li>
	<li>Check wells under microscope for uniform cell distribution and return to incubator for 24 hr.</li>
</ol>
3. GST-kinase Pulldown
<ol>
	<li>Make fresh: 4 mM pervanadate solution by mixing 60 &#181;l of 0.2 M sodium vanadate with 540 &#181;l of H2O. In a second tube mix 2.7 &#181;l of 30% peroxide and 1.4 ml of PBS. Add the two solutions together and let sit for 15 min before use.</li>
	<li>Using a multichannel pipette (do not use an automated liquid dispenser as it creates too much turbulence in the well) dispense 2 &#181;l of 0.25 M CaCl2 in each well, followed by 2.5 &#181;l of the pervanadate prepared in step 3.1. Incubate each plate at 37 &#176;C for 10 min and then place on ice.</li>
	<li>Prepare 35 ml of lysis buffer by adding DTT, PMSF, and sodium vanadate as indicated in Section 1 (Preparation of Reagents).</li>
	<li>Keeping plates on ice, remove medium from each well using a vacuum aspirator. Immediately add 50 &#181;l/well of ice cold lysis buffer using an automated liquid dispenser fitted with a standard cassette. Let sit 30 min on ice to lyse (optional: can stop here if necessary by sealing plate and storing at -80 &#176;C. To continue, thaw plates on ice).</li>
	<li>Spin plates at 1,900 x g for 3 min at 4 &#176;C.</li>
	<li>Scrape cells from each well using a multichannel pipette and transfer all contents to appropriately labeled V-bottom 96 well plates. Spin plates at 1,900 x g for 10 min at 4 &#176;C.</li>
	<li>During spin, fill glutathione coated plates (one for each 96-well plate) with 100 &#181;l/well of ice cold lysis buffer (without PMSF) as a rinse. Keep plates on ice.</li>
	<li>Remove V-bottom plates from the centrifuge. One at a time, invert the glutathione plates over a sink to shake out the lysis buffer and blot on a paper towel. Transfer lysis buffer from the V-bottom plates to the glutathione plates by tilting the plate and using a multichannel pipette, being careful to not disturb the pellet at the bottom. Cover plates and leave on ice for minimum 2 hr to bind.</li>
	<li>Close to the end of the 2 hr binding step, prepare a radioactivity workstation, ensuring the necessary safety precautions are in place for radioactive work. Set hybridization oven to 30 &#176;C.</li>
	<li>Prepare 200 ml of lysis buffer by adding DTT and sodium vanadate as indicated in Section 1. PMSF is not required at this stage.</li>
	<li>Invert the glutathione plates over a sink to shake out the lysis buffer and blot on a paper towel. Rinse wells 3x with 100 &#181;l lysis buffer (without PMSF). Do not let wells sit dry &#8212; keep them in rinse until ready to proceed.</li>
	<li>Prepare 55 ml of 1x kinase buffer (KB) by diluting the 10x stock and adding DTT as indicated in Section 1. Rinse plates once with 50 &#181;l of 1x KB using an automated liquid dispenser fitted with a standard volume cassette. Leave 1x KB in wells until solution A is ready:
	<ol>
		<li>Prepare Solution A by adding 500 to 530 &#181;g of the substrate of interest, 500 &#181;g of myelin basic protein (MBP), 2.65 ml of 10x KB, 13.25 &#181;l of 1 M DTT, and H2O up to 15.9 ml.</li>
	</ol>
	</li>
	<li>One at a time, invert the plates over a sink to remove 1x KB rinse, blot on paper towel, and immediately add 30 &#181;l of Solution A using an automated liquid dispenser fitted with a small volume cassette. Keep plates on ice.</li>
	<li>Prepare Solution B in the radioactivity work area by adding 2.5 ml 10x M-ATP, 500 &#181;Ci gamma-32P ATP, and H2O to 10 ml.
	<ol>
		<li>Add 20 &#181;l of Solution B per well using a repeater pipette which aids in mixing due to ejection force. Cover and incubate in 30 &#176;C hybridization oven for 30 min.</li>
	</ol>
	</li>
	<li>After 30 min, transfer plates back to ice. Add 50 &#181;l of 2x SDS lysis buffer to each well using a multichannel pipette. Can proceed at this point to the next step, or seal the plates with aluminum foil and store at -20 &#176;C until convenient.</li>
</ol>
4. Running, Staining, and Drying Gels
Note: All work should be performed in an area designated for radioactivity.
<ol>
	<li>Turn on hybridization oven and set to 85 &#176;C. Thaw plates at room temperature. Once oven has reached temperature, transfer plates to oven and incubate for 10 min to denature samples.</li>
	<li>Load 26-well pre-cast gels with 15 &#181;l of each reaction using a multichannel pipette to fill several wells at once. Care must be taken that all tips align with corresponding well before adding samples. Run gel at 150 V. Do not let the tracking dye (blue line) run off of the bottom of the gel as this contains the unincorporated ATP.</li>
	<li>Dismantle the gels and cut off the unincorporated ATP (blue line) using a scalpel or straight edge as it will overexpose the films. Place the gels in labeled containers and cover with Coomassie stain for 15 min.</li>
	<li>Remove the Coomassie stain, briefly rinse the gels with water, and add destain solution. Destain the gels until the proteins are clearly visible. A band for MBP and a band for the substrate should be visible for each sample.</li>
	<li>To dry the gels:
	<ol>
		<li>Cut a large sheet of filter paper and place it on the dryer.</li>
		<li>Wet a cellophane sheet in distilled water until it is smooth and wrinkle free, and place it on top of the paper.</li>
		<li>Lay the gels on top of the cellophane sheet, making a note of the order of gels. Wet a second cellophane sheet and place on top of the gels.</li>
		<li>Roll out all bubbles (a plate sealing roller works well for this) for a nice uniform surface. Close the flap, turn on the vacuum, and dry the gels for 3 hr at 80 &#176;C.</li>
	</ol>
	</li>
	<li>Once gels are dry, expose them to XAR film using a screen to intensify the signal. Wrap the cassette with saran wrap or a plastic bag and seal with tape to keep out frost. Store the cassette at -80 &#176;C overnight.</li>
</ol>
5. Developing XAR Films
<ol>
	<li>The following day, remove cassette from freezer and let thaw at room temperature. Develop the film in a darkroom using a film processor according to manufacturer&#8217;s instructions. 
	Note: Examine the films for evidence of kinase-substrate pairs. A second longer exposure can also be useful to detect weaker phosphorylation events.</li>
</ol>

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	<li>Meisenhelder, J., Hunter, T., van der Geer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphopeptide+mapping+and+identification+of+phosphorylation+sites.">Phosphopeptide mapping and identification of phosphorylation sites.</a> Curr Protoc Mol Biol. 18, Unit 18 19 (2001).</li><li>Doll, S., Burlingame, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+detection+and+assignment+of+protein+posttranslational+modifications.">Mass spectrometry-based detection and assignment of protein posttranslational modifications.</a> ACS chem. 10, 63-71 (2015).</li><li>Sharma, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultradeep+human+phosphoproteome+reveals+a+distinct+regulatory+nature+of+Tyr+and+Ser/Thr-based+signaling.">Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling.</a> Cell rep. 8, 1583-1594 (2014).</li><li>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=The+regulation+of+protein+function+by+multisite+phosphorylation--a+25+year+update.">The regulation of protein function by multisite phosphorylation--a 25 year update.</a> Trends Biochem Sci. 25, 596-601 (2000).</li><li>Walsh, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Posttranslation Modification of Proteins: Expanding Nature's Inventory. , (2006).</li><li>Boersema, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+qualitative+and+quantitative+profiling+of+tyrosine+phosphorylation+using+a+combination+of+phosphopeptide+immunoaffinity+purification+and+stable+isotope+dimethyl+labeling.">In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling.</a> Mol Cell Proteomics. 9, 84-99 (2010).</li><li>Hutti, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+determining+protein+kinase+phosphorylation+specificity.">A rapid method for determining protein kinase phosphorylation specificity.</a> Nat Methods. 1, 27-29 (2004).</li><li>Johnson, S. A., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinomics:+methods+for+deciphering+the+kinome.">Kinomics: methods for deciphering the kinome.</a> Nat Methods. 2, 17-25 (2005).</li><li>Pawson, T., Nash, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+cell+regulatory+systems+through+protein+interaction+domains.">Assembly of cell regulatory systems through protein interaction domains.</a> Science. 300, 445-452 (2003).</li><li>Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+yeast+protein+kinases+using+protein+chips.">Analysis of yeast protein kinases using protein chips.</a> Nat Genet. 26, 283-289 (2000).</li><li>Shah, K., Shokat, K. 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+chemical+genetic+approach+for+the+identification+of+direct+substrates+of+protein+kinases.">A chemical genetic approach for the identification of direct substrates of protein kinases.</a> Methods Mol Biol. 233, 253-271 (2003).</li><li>Zou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKIS:+computational+identification+of+protein+kinases+for+experimentally+discovered+protein+phosphorylation+sites.">PKIS: computational identification of protein kinases for experimentally discovered protein phosphorylation sites.</a> BMC bioinform. 14, 247 (2013).</li><li>Varjosalo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+active+and+kinase-deficient+kinome+collection+for+identification+of+kinases+regulating+hedgehog+signaling.">Application of active and kinase-deficient kinome collection for identification of kinases regulating hedgehog signaling.</a> Cell. 133, 537-548 (2008).</li><li>Jansson, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+controls+CREB+activity+in+islet+cells+via+regulated+phosphorylation+of+TORC2.">Glucose controls CREB activity in islet cells via regulated phosphorylation of TORC2.</a> Proc Natl Acad Sci U S A. 105, 10161-10166 (2008).</li><li>Fu, A., Screaton, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+kinomics+to+delineate+signaling+pathways:+control+of+CRTC2/TORC2+by+the+AMPK+family.">Using kinomics to delineate signaling pathways: control of CRTC2/TORC2 by the AMPK family.</a> Cell Cycle. 7, 3823-3828 (2008).</li><li>Abu-Thuraia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axl+phosphorylates+elmo+scaffold+proteins+to+promote+rac+activation+and+cell+invasion.">Axl phosphorylates elmo scaffold proteins to promote rac activation and cell invasion.</a> Mol Cell Biol. 35, 76-87 (2015).</li><li>Manning, G., Whyte, D. 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The authors have nothing to disclose.

Since the original publications describing the approach 14,15, the original library of 180 GST-kinases has been expanded to 420 members, or ~80% of the human protein kinome. With the expanded library, the protocol as described takes 4-5 days and then 1-4 days to develop films (as deemed necessary), which could be shortened by use of phosphorimaging and digital signal enhancement. There are several key steps where care must be taken (See Figure 1 for overview of protocol). First, is the health ...

Protein post-translational modifications (PTMs) are essential for intracellular communication. Perhaps the best studied of all PTMs is phosphorylation, catalyzed by protein kinases, which regulate a myriad of protein functions, including their biochemical activity, subcellular localization, conformation, and stability. The identification of phosphorylation sites on target proteins can be accomplished by tryptic phosphopeptide mapping or by now-standard proteomic techniques using samples enriched for phosphorylated peptides 1,2. While three quarters of the expressed proteome are expected to be phosphorylated 3 and an identified 200,000 phosphorylation sites 5, with estimates up to 1 million 6, many of these have no assigned biology, signaling pathway, or protein kinase.
While identification of phosphorylated sites is relatively straightforward, a comparatively greater challenge is to identify the cognate kinase(s) that targets these sites, a process we refer to as mapping kinase:substrate pairs. Several approaches for identifying kinase:substrate pairs have been described, either starting with a kinase of interest and looking for its substrates or starting with a substrate of interest and attempting to find a modifying kinase experimentally 7-11 or computationally 12. To identify kinases for a known phosphorylated substrate, bioinformatics can be used to identify proteins that contain a short conserved sequence of amino acids flanking the phosphorylated residue (the consensus site), as well as identifying kinases that form a precipitable complex with the substrate. However, these approaches are time-consuming and often do not meet with success.
We developed a systematic functional approach to rapidly identify kinases that can phosphorylate a given substrate 13. The screen assay produces excellent specificity, with very clear selection for potential cognate kinases. Given the centrality of phosphorylation to biological signaling, the screen is useful for discovery in virtually all cell signaling pathways 14-16. The screen involves performing a large-scale kinase assay with a library of human protein kinases. The kinases have been tagged with bacterial glutathione S-transferase (GST) protein and are purified from mammalian cell extracts, which means that the recombinant enzymes &#8212; unlike those prepared from bacteria &#8212; are generated in the presence of the upstream protein kinases often required for the recombinant enzymes to have activity in vitro. Indeed, while serine, threonine, and tyrosine kinase activity required for downstream kinase activation are present in yeast 10, the yeast genome encodes 122 protein kinases, indicating that the mammalian kinome, with over 500 genes 17, has become significantly more complex in order to regulate the processes unique to higher order organisms. Moreover, the effect of different stimuli relevant to cell biology and human disease (such as small molecules, growth factors, hormones, etc.) can be used to 14,15modulate kinase activity in an appropriate context.

<table><tbody><tr><td>Lysis buffer</td><td>Made in house</td><td></td><td>See Protocol step 1.1</td></tr><tr><td>10x kinase buffer</td><td>Made in house</td><td></td><td>See Protocol step 1.2</td></tr><tr><td>10x M-ATP</td><td>Made in house</td><td></td><td>See Protocol step 1.3</td></tr><tr><td>Human kinase plasmids</td><td>Orfeome, Invitrogen, Origene</td><td></td><td>GST-tagged in house</td></tr><tr><td>96 well plates</td><td>Fisher Scientific</td><td>CS003595</td><td></td></tr><tr><td>293T cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>DMEM</td><td>Fisher Scientific</td><td>SH3002201</td><td>supplement with 100&amp;nbsp;U/ml penicillin, 100&amp;nbsp;&amp;#956;g/ml streptomycin, 10% fetal calf serum.</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Sanyo</td><td>MCO-17AIC</td><td></td></tr><tr><td>15 cm cell culture dishes</td><td>Fisher Scientific</td><td>877224</td><td></td></tr><tr><td>Reduced serum medium</td><td>Invitrogen</td><td>22600-050</td><td></td></tr><tr><td>Lipid-based transfection reagent</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>Automated liquid dispenser</td><td>Thermo Scientific</td><td>5840300</td><td></td></tr><tr><td>Small cassette attachment</td><td>Thermo Scientific</td><td>24073295</td><td></td></tr><tr><td>Standard cassette attachment</td><td>Thermo Scientific</td><td>14072670</td><td></td></tr><tr><td>4 mM pervanadate</td><td>Made in house</td><td></td><td>See Protocol step 3.1</td></tr><tr><td>0.25 M CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Made in house</td><td></td><td></td></tr><tr><td>Multichannel pipette (20-200 &amp;#956;l)</td><td>Labnet</td><td>p4812-200</td><td></td></tr><tr><td>Multichannel pipette (1-10 &amp;#956;l)</td><td>Thermo Scientific</td><td>4661040</td><td></td></tr><tr><td>V-bottom 6-well plates</td><td>Evergreen Scientific</td><td>290-8116-01V</td><td></td></tr><tr><td>Glutathione coated 96-well plates</td><td>Fisher Scientific</td><td>PI-15240</td><td></td></tr><tr><td>Hybridization oven</td><td>Biostad</td><td>350355</td><td></td></tr><tr><td>GST tagged substrate</td><td>Made in house</td><td></td><td></td></tr><tr><td>Myelin Basic Protein (MBP)</td><td>Sigma</td><td>M1891</td><td></td></tr><tr><td>Repeater pipette (1 ml)</td><td>Eppendorf</td><td>22266209</td><td></td></tr><tr><td>&lt;sup&gt;32&lt;/sup&gt;P gamma-ATP</td><td>Perkin Elmer</td><td>BLU502Z500UC</td><td></td></tr><tr><td>2x SDS lysis buffer (100&amp;nbsp;ml)</td><td>Made in house</td><td></td><td>See Protocol step 1.4</td></tr><tr><td>26-well precast TGX gels</td><td>BioRad</td><td>567-1045</td><td>gel percentage required is dependent on the molecular weight of the substrate of interest</td></tr><tr><td>Coomassie stain</td><td>Made in house</td><td></td><td>0.1% Coomassie R250, 10% acetic acid, 40% methanol</td></tr><tr><td>Coomassie destain</td><td>Made in house</td><td></td><td>10% acetic acid, 20% methanol</td></tr><tr><td>Labeled gel containers</td><td>Made in house</td><td></td><td>Used plastic lids from empty tip boxes, just big enough to contain one gel</td></tr><tr><td>Whatman filter paper</td><td>Fisher Scientific</td><td>57144</td><td></td></tr><tr><td>Cellophane sheets (2)</td><td>BioRad</td><td>165-0963</td><td></td></tr><tr><td>Gel dryer</td><td>Labconco</td><td>4330150</td><td></td></tr><tr><td>Double emulsion autoradiography film</td><td>VWR</td><td>IB1651454</td><td></td></tr><tr><td>Film cassette</td><td>Fisher Scientific</td><td>FBAC-1417</td><td></td></tr><tr><td>Intensifying screen</td><td>Fisher Scientific</td><td>FBIS-1417</td><td></td></tr><tr><td>Plate sealing rubber roller</td><td>Sigma</td><td>R1275</td><td></td></tr></tbody></table>

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.

Representative results from a screen are shown in Figure 2. 180 kinases were screened using a GST-tagged peptide substrate corresponding to aa 268-283 from CRTC2 as well as the classic kinase assay substrate myelin basic protein (MBP). Only two kinases, MARK2 and the highly related kinase MARK3 phosphorylated the CRTC2 peptide. MBP is included as an internal control in all assays, as it contains many phosphorylatable residues and runs at 18 kDa, toward the bottom of the gel. This allows for an interpreta...

Watch this Scientific Journal Video about Identification of Kinase-substrate Pairs Using High Throughput Screening at JoVE.com

Identification of Kinase-substrate Pairs Using High Throughput Screening

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 ...

identification-kinase-substrate-pairs-using-high-throughput

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JoVE Journal

Biology

8.1K Views.  Children's Hospital of Eastern Ontario Research Institute. 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.

Video: Identification of Kinase-substrate Pairs Using High Throughput Screening

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 ...

Biochemistry

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

Developmental Biology

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

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 ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

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 ...

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Bioengineering

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

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