This work was supported in part by a Commonwealth Universal Research Enhancement grant from the Pennsylvania Department of Health to T.I.S.

NOTE: Ensure that the required materials are available and properly prepared (see Table of Materials).
1. Preparation
<ol>
	<li>Mechanically lyse tissue sample (1-2 mg of tissue in 100 &#181;L of lysis buffer, Table 1) or cultured cells (10 cm plate that is 80-90% confluent in 350 &#181;L of lysis buffer), with the goal being to collect a total of 900 &#181;L of sample for the experiment. Note that this volume is in slight excess of what is required for the experiment described below.</li>
	<li>Spin down the samples by centrifugation at 17,500 x g for 3 minutes at 4 &#176;C. Upon completion, transfer 270 &#181;L of the supernatant to each of three new 1.7 mL microcentrifuge tubes. There will be approximately 90 &#181;L remaining. Remove 40 &#181;L to be used as an &#8220;input control&#8221; and place in a new tube. Place all samples on ice.</li>
</ol>
2. Kinase assay: thiophosphorylation and alkylation
<ol>
	<li>Label the three tubes containing 270 &#181;L each as follows: &#8220;kinase rxn&#8221;, &#8220;GTP&#947;S only&#8221;, and &#8220;PNBM (p-nitrobezyl mesylate) only&#8221;. Prepare the kinase reactions.
	<ol>
		<li>To the &#8220;kinase rxn&#8221; tube, add 2.7 &#181;L (equivalent to 1,350 U) of CK2, then add 2.7 &#181;L of 2.5 mM GTP&#947;S.</li>
		<li>To the &#8220;GTP&#947;S only&#8221; tube, add 2.7 &#181;L of 2.5 mM GTP&#947;S, and then add 2.7 &#181;L of lysis buffer.</li>
		<li>To the &#8220;PNBM only&#8221; tube, add 5.4 &#181;L of lysis buffer. Flick all tubes to mix and then immediately place on ice.</li>
	</ol>
	</li>
	<li>Incubate all three tubes for 1 min in a 30 &#176;C water bath. Following incubation, add 13.5 &#181;L of 12 mg/mL PNBM to all three tubes. Invert to mix samples. Incubate these samples at room temperature for 1 h.</li>
	<li>After starting the incubation in step 2.2, prepare the desalting columns as soon as possible as the process takes approximately 45 min.</li>
</ol>
3. Preparation of desalting columns
<ol>
	<li>Initially prepare columns (3 for this example experiment) by inverting each column several times to re-suspend the Sephadex G-25 resin in the storage buffer. Allow the resin to settle by attaching each column to a clamp stand and letting it sit undisturbed for approximately 5 min.</li>
	<li>Following settling of the Sephadex G-25 resin, remove the caps from both the top and bottom of the column to allow the storage buffer to drain by gravity and have a tube placed below the bottom opening to collect the flow-through for discard.</li>
	<li>Once storage buffer is depleted, add approximately 2.7 mL of lysis buffer in order to equilibrate the columns. Collect the flow-through and discard. Repeat 3 times. Following the final equilibration, the columns are ready for step 4.1.</li>
</ol>
4. Removal of PNBM
<ol>
	<li>After the 1 h incubation (step 2.2) and column preparation (step 3) are complete, apply the samples to the columns. Label each column as follows: &#8220;kinase rxn&#8221;, &#8220;GTP&#947;S only&#8221;, and &#8220;PNBM only&#8221;. Load all of each sample onto its respective column. Collect and discard the flow-through.</li>
	<li>Wash samples by adding 420 &#181;L of lysis buffer to each column. Allow lysis buffer to filter through the column and collect the flow-through for discard. Following this wash step, place tubes in position for collection of samples.</li>
	<li>Elute samples by adding 500 &#181;L of lysis buffer to each column. Collect the flow-through that now contains thiophosphorylated and alkylated CK2 substrates.</li>
</ol>
5. Immunoprecipitation: Part I
<ol>
	<li>Prepare samples for immunoprecipitation by first removing 80 &#181;L for an &#8220;elution input control&#8221; sample from each of the respective elutions (&#8220;kinase rxn&#8221;, &#8220;GTP&#947;S only&#8221;, and &#8220;PNBM only&#8221;) collected in step 4.3. Following removal of 80 &#181;L from each sample, there will be approximately 420 &#181;L remaining per sample.
	<ol>
		<li>Split each sample into 2 tubes containing 200 &#181;L each. Label each respective tube: &#8220;kinase rxn anti-thiophosphate ester&#8221;, &#8220;kinase rxn IgG&#8221;, &#8220;GTP&#947;S anti-thiophosphate ester&#8221;, &#8220;GTP&#947;S IgG&#8221;, &#8220;PNBM anti-thiophosphate ester&#8221;, and &#8220;PNBM IgG&#8221;.</li>
	</ol>
	</li>
	<li>Add 2.8 &#181;g of anti-thiophosphate ester antibody to each of the anti-thiophosphate ester-labeled tubes and 2.8 &#181;g of isotype control antibody to each of the IgG-labeled tubes. Place tubes on a rotator at 4 &#176;C for 2 h.</li>
	<li>Begin preparation of protein A/G bead during the last 15 min of the 2 h incubation in step 5.2.</li>
</ol>
6. Protein A/G agarose bead preparation
<ol>
	<li>Briefly vortex the storage tube to ensure beads are completely re-suspended. Cut off the end of a P200 pipette tip using a clean razor blade in order to increase gauge size. Pipet 100 &#181;L of the bead slurry per immunoprecipitation into a new 1.7 mL microcentrifuge tube. For this example, a total of 6 tubes are needed.</li>
	<li>Centrifuge the tubes at 17,500 x g for 1 min at 4 &#176;C. Remove the supernatant and discard. Re-suspend the beads in 200 &#181;L of lysis buffer and briefly vortex. Repeat the spin and wash steps 3 times.</li>
	<li>Following the final wash, place the beads on ice until the incubation in step 5.2 is complete.</li>
</ol>
7. Immunoprecipitation: Part II
<ol>
	<li>After the 2 h incubation from step 5.2, spin down the samples at 17,500 x g for 3 min at 4 &#176;C. Following centrifugation add 200 &#181;L from each sample to the tubes with the washed beads. Place the tubes on a rotator at 4 &#176;C for 1 h.</li>
	<li>Following the 1 h incubation, centrifuge the tubes at 17,500 x g for 1 min at 4 &#176;C. Next remove 40 &#181;L of supernatant from each sample and save as a &#8220;depletion control&#8221; (total 6 tubes). Remove the remainder of the supernatant and discard. Take care not to disturb the beads.</li>
	<li>Wash the samples by adding 200 &#181;L of lysis buffer and vortexing briefly. Then centrifuge at 17,500 x g for 1 min at 4 &#176;C. Remove the supernatant and discard. Repeat the wash and spin steps 3 times. Take care not to disturb the beads during these steps.</li>
	<li>After completing the wash steps, add 50 &#181;L of 2x sample buffer to each sample containing beads. For all other samples, add 8 &#181;L of 6x sample buffer: &#8220;input control&#8221;, &#8220;elution input controls&#8221;, and &#8220;depletion controls&#8221;.</li>
	<li>Once buffer is added to the tubes, pipet up and down to mix, and heat all samples at 95 &#176;C for 5 min before proceeding with SDS-PAGE.</li>
</ol>
8. Analysis/Validation of results
<ol>
	<li>Validate successful CK2-dependent thiophosphorylation and alkylation.
	<ol>
		<li>To determine the efficacy of CK2-mediated thiophosphorylation in step 2.2, perform SDS-PAGE and Western blotting by running 15-20 &#181;L of the &#8220;elution input controls&#8221; collected in step 5.1 on a 12.5% polyacrylamide gel.</li>
		<li>Probe membranes with the following antibodies: anti-thiophosphate ester, anti-CK2&#945;, and anti-GAPDH (or other appropriate loading control). If this step was successful, an enhanced anti-thiophosphate ester signal should be apparent in the &#8220;kinase rxn&#8221; lane compared to the other two lanes (Figure 2).</li>
	</ol>
	</li>
	<li>Visualize enriched putative substrates of CK2 and determine protein identity.
	<ol>
		<li>To assess if the immunoprecipitation steps were successful, run 25-30 &#181;L of the samples eluted from the beads in step 7.4 on a separate 12.5% polyacrylamide gel. Ensure that all equipment is clean and wear gloves at all times during this step to minimize contamination.</li>
		<li>Stain the gel with Coomassie blue to visualize enriched proteins from various stages of the experimental protocol (Figure 3). Using new razorblades, carefully excise unique bands present in the &#8220;kinase rxn anti-thiophosphate ester IP&#8221; lane, noting their approximate molecular weights.</li>
		<li>Submit these bands for protein identification by liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Figure 4). If antibodies directed against the identified proteins are available, confirm the results of mass spectrometry by SDS-PAGE and immunoblotting of input, depleted, and IP fractions collected during the course of the protocol (Figure 4).</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-thousand-and-one+substrates+of+protein+kinase+CK2.">One-thousand-and-one substrates of protein kinase CK2.</a> The FASEB Journal. 17 (3), 349-368 (2003).</li><li>Niefind, K., Guerra, B., Ermakowa, I., Issinger, O. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+human+protein+kinase+CK2:+insights+into+basic+properties+of+the+CK2+holoenzyme.">Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme.</a> The EMBO Journal. 20 (19), 5320-5331 (2001).</li><li>Sarno, S., Ghisellini, P., Pinna, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+activation+mechanism+of+protein+kinase+CK2.+The+N-terminal+segment+is+essential+for+constitutive+activity+of+the+catalytic+subunit+but+not+of+the+holoenzyme.">Unique activation mechanism of protein kinase CK2. The N-terminal segment is essential for constitutive activity of the catalytic subunit but not of the holoenzyme.</a> Journal of Biological Chemistry. 277 (25), 22509-22514 (2002).</li><li>Niefind, K., Putter, M., Guerra, B., Issinger, O. G., Schomburg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTP+plus+water+mimic+ATP+in+the+active+site+of+protein+kinase+CK2.">GTP plus water mimic ATP in the active site of protein kinase CK2.</a> Nature Structural &amp; Molecular Biology. 6 (12), 1100-1103 (1999).</li><li>Lou, D. Y., 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=The+alpha+catalytic+subunit+of+protein+kinase+CK2+is+required+for+mouse+embryonic+development.">The alpha catalytic subunit of protein kinase CK2 is required for mouse embryonic development.</a> Molecular and Cellular Biology. 28 (1), 131-139 (2008).</li><li>Buchou, T., 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=Disruption+of+the+regulatory+beta+subunit+of+protein+kinase+CK2+in+mice+leads+to+a+cell-autonomous+defect+and+early+embryonic+lethality.">Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality.</a> Molecular and Cellular Biology. 23 (3), 908-915 (2003).</li><li>Chua, M. 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=CK2+in+Cancer:+Cellular+and+Biochemical+Mechanisms+and+Potential+Therapeutic+Target.">CK2 in Cancer: Cellular and Biochemical Mechanisms and Potential Therapeutic Target.</a> Pharmaceuticals (Basel). 10 (1), (2017).</li><li>Tawfic, S., 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=Protein+kinase+CK2+signal+in+neoplasia.">Protein kinase CK2 signal in neoplasia.</a> Histology and Histopathology. 16 (2), 573-582 (2001).</li><li>Hanif, I. M., Hanif, I. M., Shazib, M. A., Ahmad, K. A., Pervaiz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Casein+Kinase+II:+an+attractive+target+for+anti-cancer+drug+design.">Casein Kinase II: an attractive target for anti-cancer drug design.</a> The International Journal of Biochemistry &amp; Cell Biology. 42 (10), 1602-1605 (2010).</li><li>Siddiqui-Jain, 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=CX-4945,+an+orally+bioavailable+selective+inhibitor+of+protein+kinase+CK2,+inhibits+prosurvival+and+angiogenic+signaling+and+exhibits+antitumor+efficacy.">CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy.</a> Cancer Research. 70 (24), 10288-10298 (2010).</li><li>Chon, H. J., Bae, K. J., Lee, Y., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+casein+kinase+2+inhibitor,+CX-4945,+as+an+anti-cancer+drug+in+treatment+of+human+hematological+malignancies.">The casein kinase 2 inhibitor, CX-4945, as an anti-cancer drug in treatment of human hematological malignancies.</a> Frontiers in Pharmacology. 6, 70 (2015).</li><li>Perea, S. 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=CIGB-300,+a+novel+proapoptotic+peptide+that+impairs+the+CK2+phosphorylation+and+exhibits+anticancer+properties+both+in+vitro+and+in+vivo.">CIGB-300, a novel proapoptotic peptide that impairs the CK2 phosphorylation and exhibits anticancer properties both in vitro and in vivo.</a> Molecular and Cellular Biochemistry. (1-2), 163-167 (2008).</li><li>Xiao, S., 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=Induced+expression+of+nucleolin+phosphorylation-deficient+mutant+confers+dominant-negative+effect+on+cell+proliferation.">Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation.</a> PLoS One. 9 (10), e109858 (2014).</li><li>Shi, Y., Brown, E. D., 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=Expression+of+recombinant+human+casein+kinase+II+and+recombinant+heat+shock+protein+90+in+Escherichia+coli+and+characterization+of+their+interactions.">Expression of recombinant human casein kinase II and recombinant heat shock protein 90 in Escherichia coli and characterization of their interactions.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (7), 2767-2771 (1994).</li><li>Mollapour, M., Tsutsumi, S., Kim, Y. 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P., 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=Protein+kinase+CK2+is+a+constitutively+active+enzyme+that+promotes+cell+survival:+strategies+to+identify+CK2+substrates+and+manipulate+its+activity+in+mammalian+cells.">Protein kinase CK2 is a constitutively active enzyme that promotes cell survival: strategies to identify CK2 substrates and manipulate its activity in mammalian cells.</a> Methods in Enzymology. , 471-493 (2010).</li><li>Rusin, S. F., Adamo, M. E., Kettenbach, A. 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The authors have nothing to disclose.

Here, we describe a relatively simple biochemical method for identifying substrates of protein kinase CK2 from a complex biological sample. The critical steps of this protocol are based on the unusual enzymatic properties of CK2 and include CK2-dependent thiophosphorylation of specific substrate proteins using GTP&#947;S and their subsequent immunoprecipitation and identification. With these results, we have demonstrated the utility and versatility of this approach as we have now applied this strategy in both hu...

Protein kinases are key components of signal transduction cascades. Phosphorylation of substrate proteins by these enzymes elicits biological responses that regulate critical events controlling cell division, metabolism, and differentiation, among others. CK2 is a ubiquitously expressed, acidophilic serine/threonine kinase that is conserved from yeast to humans and that plays important roles in many cellular processes ranging from transcriptional regulation to cell cycle progression to apoptosis1,2,3. The enzyme is a heterotetramer composed of two catalytic &#945; (or &#945;&#39;) subunits and two regulatory &#946; subunits4. In addition to being highly pleiotropic, CK2 exhibits two other unusual characteristics that complicate its analysis, namely constitutive activity5 and dual co-substrate specificity6. This latter property endows CK2 with the ability to use GTP as well as ATP for phosphorylation of substrate proteins.
Genetic deletion of the catalytic or regulatory subunits of CK2 in mice results in embryonic lethality indicating that it plays crucial roles during development and organogenesis7,8. CK2 is also overexpressed in several types of cancer and thus represents a promising therapeutic target9,10,11. Indeed, specific inhibitors that target CK2 kinase activity are currently under investigation for this purpose12,13,14. While inhibition of CK2 is a viable option, given its pleiotropic nature, an alternative and perhaps more rational approach would be to target critical CK2 substrates that underlie the progression of certain cancers. Therefore, the comprehensive identification and characterization of CK2 substrate proteins would be of significant benefit for elucidating the specific function(s) of this kinase within a particular tissue or tumor type.
Here, we describe a versatile biochemical method for identifying CK2 substrates from a complex biological sample such as a cell or tissue lysate. This protocol takes advantage of the dual co-substrate specificity of CK2 by use of the GTP analogue GTP&#947;S (guanosine 5&#39;-[&#947;-thio]triphosphate) that other endogenous kinases cannot use. This effectively allows the kinase to &#34;label&#34; its substrates within this sample for subsequent isolation and identification.

<table><tbody><tr><td>12 mg/mL PNBM</td><td>Abcam</td><td>ab138910</td><td>40.5 &amp;micro;L</td></tr><tr><td>2.5 mM GTP&amp;gamma;S</td><td>Sigma-Aldrich</td><td>G8634-1MG</td><td>5.4 &amp;micro;L</td></tr><tr><td>Anti-CK2&amp;alpha; (E-7) mouse monoclonal antibody</td><td>Santa Cruz Biotechnology</td><td>sc-373894</td><td>1:1000 for Western blotting</td></tr><tr><td>Anti-GAPDH (6C5) mouse monoclonal antibody</td><td>Santa Cruz Biotechnology</td><td>sc-32233</td><td>1:1000 for Western blotting</td></tr><tr><td>Anti-nucleolin rabbit polyclonal antibody</td><td>Abcam</td><td>ab22758</td><td>1:1000 for Western blotting</td></tr><tr><td>Anti-thiophosphate ester [51-8] rabbit monoclonal antibody</td><td>Abcam</td><td>ab92570</td><td>Varies (final concentration 2.8 &amp;micro;g for each sample)</td></tr><tr><td>Centrifuge pre-set to 4&amp;ordm;C</td><td>ThermoScientific</td><td>Sorvall Legend Micro 21R Cat# 75-772-436&amp;nbsp;</td><td></td></tr><tr><td>cOmplete Mini EDTA-Free Protease Inhibitor</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>Lysis Buffer</td><td>See recipe below</td><td>See recipe below</td><td>30 mL</td></tr><tr><td>Normal rabbit IgG antibody (isotype control)</td><td>Cell Signaling Technology</td><td>2729S&amp;nbsp;</td><td>Varies (final concentration 2.8 &amp;micro;g for each sample)</td></tr><tr><td>PD MiniTrap Column</td><td>GE Healthcare</td><td>28-9180-10</td><td>3 columns</td></tr><tr><td>Protein A/G Plus Agarose Beads</td><td>Santa Cruz Biotechnology</td><td>sc-2003</td><td>600 &amp;micro;L</td></tr><tr><td>Recombinant human CK2 holoenzyme</td><td>New England Biolabs</td><td>P6010S</td><td>2.7 &amp;micro;L</td></tr><tr><td>Rotator</td><td>Labnet: Mini Labroller</td><td>Mini Labroller SKU# H5500</td><td></td></tr><tr><td>T98G human glioblastoma cells</td><td>ATCC</td><td>CRL-1690</td><td></td></tr><tr><td>Water bath pre-set to 30&amp;ordm;C</td><td>Shel Lab</td><td>H20 Bath Series Model# SWB15</td><td></td></tr></tbody></table>

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.

A schematic diagram of the experimental procedure is provided in Figure 1. The underlying basis of the technique is the unusual ability of CK2 to use GTP for phosphoryl group transfer. Addition of exogenous CK2 holoenzyme along with the GTP analogue, GTP&#947;S, to a cell lysate results in thiophosphorylation of endogenous CK2 substrates. Subsequent treatment of the lysate with the alkylating reagent p-nitrobenzyl mesylate (PNBM) generates a thiophos...

Watch this Scientific Journal Video about Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach at JoVE.com

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

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 ...

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7.3K Views.  Drexel University College of Medicine. 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.

Video: Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

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.

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 ...

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

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 ...

Biology

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 ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

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High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Assaying Protein Kinase Activity with Radiolabeled ATP

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

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

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

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Assaying the Kinase Activity of LRRK2 in vitro

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

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays