Name: identification of novel ck2 kinase substrates using a versatile biochemical approach
Uploaded: 2019-02-21T14:30:00
Description: Watch this Scientific Journal Video about Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach at JoVE.com

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 ...

<ol>
	<li>Litchfield, D. W. <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:+structure,+regulation+and+role+in+cellular+decisions+of+life+and+death.">Protein kinase CK2: structure, regulation and role in cellular decisions of life and death.</a> Biochemical Journal. 369 (Pt 1), 1-15 (2003).</li><li>Ahmed, K., Gerber, D. A., Cochet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Joining+the+cell+survival+squad:+an+emerging+role+for+protein+kinase+CK2).">Joining the cell survival squad: an emerging role for protein kinase CK2).</a> Trends in Cell Biology. 12 (5), 226-230 (2002).</li><li>Meggio, F., 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=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. S., Trepel, J., Neckers, L. <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+2+phosphorylation+of+Hsp90+threonine+22+modulates+chaperone+function+and+drug+sensitivity.">Casein kinase 2 phosphorylation of Hsp90 threonine 22 modulates chaperone function and drug sensitivity.</a> Oncotarget. 2 (5), 407-417 (2011).</li><li>McMillan, E. 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=The+protein+kinase+CK2+substrate+Jabba+modulates+lipid+metabolism+during+Drosophila+oogenesis.">The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis.</a> Journal of Biological Chemistry. 293 (8), 2990-3002 (2018).</li><li>Turowec, J. 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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Candidate+Casein+Kinase+2+Substrates+in+Mitosis+by+Quantitative+Phosphoproteomics.">Identification of Candidate Casein Kinase 2 Substrates in Mitosis by Quantitative Phosphoproteomics.</a> Frontiers in Cell and Developmental Biologyl. 5, 97 (2017).</li><li>Bian, 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=Global+screening+of+CK2+kinase+substrates+by+an+integrated+phosphoproteomics+workflow.">Global screening of CK2 kinase substrates by an integrated phosphoproteomics workflow.</a> Scientific Reports. 3, 3460 (2013).</li><li>Franchin, C., 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=Quantitative+analysis+of+a+phosphoproteome+readily+altered+by+the+protein+kinase+CK2+inhibitor+quinalizarin+in+HEK-293T+cells.">Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2 inhibitor quinalizarin in HEK-293T cells.</a> Biochimica et Biophysica Acta. 1854 (6), 609-623 (2015).</li><li>Franchin, C., 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=Re-evaluation+of+protein+kinase+CK2+pleiotropy:+new+insights+provided+by+a+phosphoproteomics+analysis+of+CK2+knockout+cells.">Re-evaluation of protein kinase CK2 pleiotropy: new insights provided by a phosphoproteomics analysis of CK2 knockout cells.</a> Cellular and Molecular Life Sciences. 75 (11), 2011-2026 (2018).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" style="width:2010px;" width="2008">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">12 mg/mL PNBM</td>
			<td style="width:599px;">Abcam</td>
			<td style="width:599px;">ab138910</td>
			<td style="width:406px;">40.5 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.5 mM GTP&#947;S</td>
			<td>Sigma-Aldrich</td>
			<td>G8634-1MG</td>
			<td>5.4 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CK2&#945; (E-7) mouse monoclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-373894</td>
			<td>1:1000 for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Anti-nucleolin rabbit polyclonal antibody</td>
			<td>Abcam</td>
			<td style="width:599px;">ab22758</td>
			<td>1:1000 for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-thiophosphate ester [51-8] rabbit monoclonal antibody</td>
			<td>Abcam</td>
			<td style="width:599px;">ab92570</td>
			<td>Varies (final concentration 2.8 &#181;g for each sample)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge pre-set to 4&#186;C</td>
			<td>ThermoScientific</td>
			<td>Sorvall Legend Micro 21R Cat# 75-772-436&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cOmplete Mini EDTA-Free Protease Inhibitor</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysis Buffer</td>
			<td>See recipe below</td>
			<td>See recipe below</td>
			<td>30 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal rabbit IgG antibody (isotype control)</td>
			<td>Cell Signaling Technology</td>
			<td>2729S&#160;</td>
			<td>Varies (final concentration 2.8 &#181;g for each sample)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD MiniTrap Column</td>
			<td>GE Healthcare</td>
			<td>28-9180-10</td>
			<td>3 columns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein A/G Plus Agarose Beads</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2003</td>
			<td>600 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant human CK2 holoenzyme</td>
			<td>New England Biolabs</td>
			<td>P6010S</td>
			<td>2.7 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotator</td>
			<td>Labnet: Mini Labroller</td>
			<td>Mini Labroller SKU# H5500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T98G human glioblastoma cells</td>
			<td>ATCC</td>
			<td>CRL-1690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath pre-set to 30&#186;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 ...

This protocol allows for the specific labeling and subsequent enrichment and identification of endogenous CK2 substrates from a complex biological sample such as a cell or tissue lysate. This method can be applied to any cell type or tissue. Thus facilitating, the study of CK2 in various biological contexts.Demonstrating the procedure will be John Chojnowski, a graduate student from my laboratory. To begin, mechanically lyse the tissue sample or cultured cells as outlined in the text protocol to collect a total of 900 microliters of sample. Centrifuge at 17, 500 times gravity and at four degrees celsius for three minutes.Then, transfer 270 microliters of the supernatant to each of three new 1.7 milliliter microcentrifuge tubes. Remove 40 microliters of the remaining supernatant to be used as an input control, and transfer it to a new tube. Place all of the samples on ice.First, label each of the three sample tubes as shown here. To the Kinase Reaction tube, add 2.7 microliters of CK2 and 2.7 microliters of 2.5 millimolar GTPgammaS. Flick the tube to mix, and immediately place it on ice.To the GTPgammaS only tube, add 2.7 microliters of 2.5 millimolar GTPgammaS, and 2.7 microliters of Lysis Buffer. Flick this tube to mix, and immediately place it on ice. To the PNBM Only tube add 5.4 microliters of Lysis Buffer.Flick the tube to mix, and immediately place it on ice. Incubate all three tubes in a water bath at 30 degrees Celsius for one minute. After this, add 13.5 microliters of PNBM at 12 milligrams per milliliter to each tube.Invert the tubes to mix the samples, and incubate the samples at room temperature for one hour. While the samples are incubating, label each of the columns for the sample to be loaded, as shown here. Prepare the columns by inverting each column several times to resuspend the Sephadex G-25 resin in the storage buffer.Attach each column to a clamp stand, and let it sit undisturbed for approximately five minutes to let the resin settle. Next, place a tube below the bottom opening of each column. Remove the caps from both the top and the bottom of each column, to allow the storage buffer to drain into the tubes by gravity.Once the storage buffer is depleted, add approximately 2.7 milliliters of Lysis Buffer to each column to equilibrate them, making sure to collect and discard the flow through. Repeat this process of adding Lysis Buffer and discarding the flow through, three times. To begin, load each sample into its respective column.Collect and discard the flow through. Add 420 microliters of Lysis Buffer to each column to wash the samples. Let the Lysis Buffer filter through the column, and collect and discard the flow through.Then, place tubes into position under each column for sample collection. Add 500 microliters of Lysis Buffer to each column to elute the samples. Collect the flow through, which now contains an enriched population of thiophosphorylated and alkylated CK2 substrates in the kinase reaction.The GTPgammaS only reaction will contain substrates thiophosphorylated and alkylated by any endogenous CK2 present. The PNMB only reaction will contain background alkylated proteins. Remove 80 microliters from each Elution as an Elution Input Control sample.Next, split each sample into two tubes that each contain 200 microliters. Label each of the tubes to be either anti-thiophosphate ester or immunoglobulin G, as shown here. Add 2.8 micrograms of anti-thiophosphate ester antibody to each of the anti-thiophosphate ester tubes.Add 2.8 micrograms of Isotype Control antibody to each of the IGG tubes. Then place the tubes on a rotator at four degrees Celsius for two hours. During the last 15 minutes of the sample incubation, use a clean razor blade to cut the end off of a P200 pipette tip to increase the gauge size.Immediately prior to use, briefly vortex the storage tube containing the agarose beads. Using the cut pipette tip, transfer 100 microliters of the beads slurry per immunoprecipitation to a new 1.7 milliliter microcentrifuge tube. It is important to vortex the beads before each transfer to prevent beads from settling.Centrifuge the tubes at 17, 500 times gravity and at four degrees Celsius for one minute. Remove and discard the supernatant. Resuspend the beads by adding 200 microliters of Lysis Buffer and briefly vortexing to mix.Repeat this process of centrifuging and washing the beads three times. After the final wash, place the beads on ice until ready to proceed. When the samples have finished incubating, centrifuge them at 17, 500 times gravity and at four degrees Celsius for three minutes.Then, transfer 200 microliters of each sample to the tubes containing the washed beads. Place the tubes on a rotator at four degrees Celsius for one hour. Next, centrifuge the tubes at 17, 500 times gravity and at four degrees Celsius for one minute.Remove 40 microliters from each supernatant to save as a depletion control. Remove and discard the remainder of the supernatant, being careful not to disturb the beads. Wash the samples by adding 200 microliters of Lysis Buffer to each and vortexing briefly.Centrifuge at 17, 500 times gravity and at four degrees Celsius for one minute, discarding the supernatant. Repeat this wash and centrifugation process three times taking care to not disturb the beads. After this, add 50 microliters of 2X sample buffer to each sample containing beads.For all other samples, which include the Input Control, the Elution Input Controls and the Depletion Controls, add 8 microliters of 6X Sample Buffer. Pipette each tube up and down to mix with the exception of tubes containing beads, and heat all samples at 95 degrees Celsius for five minutes before proceeding with SDS-PAGE. To assess if the immunoprecipitation was successful, run 25 to 30 microliters of each sample that was eluded from beads on a separate 12.5%polyacrylamide gel.Stain each gel with Coomassie Blue to visualize enriched proteins from various stages of the experimental protocol. Using new, clean razor blades, carefully excise any unique bands present in the kinase reaction anti-thiophosphate ester IP Lane while making note of their approximate molecular weights. A new razor blade should be used for each unique band.The underlying basis of this technique is this technique is the unusual ability of CK2 to use GTP for phosphoryl group transfer. Addition of exogenous CK2 holoenzyme along with the GTP analog, GTPgammaS, to a cell lysate, results in a thiophosphorylation of endogenous CK2 substrates. Subsequent treatment of the lysate with the alkylating reagent p-Nitrobenzyl mesylate generates thiophosphate ester moiety on these specific substrate proteins, that can then be immunoprecipitated using an anti-thiophosphate ester antibody and ultimately identified by Mass spectrometry.In the representative positive results shown here, CK2 dependent, thiophosphorylation and subsequent alkylation were successful. As expected, an enhanced anti-thiophosphate ester signal by Western blotting is observed only in the lane containing the complete kinase reaction, and not in the GTPgammaS only and PNMB only treated samples. A Coomassie Blue stained gel of the immunoprecipitated and alluded proteins, also demonstrate a positive result.As multiple unique bands are evident only in the anti-thiophosphate ester IP Lane, in which the lysate was incubated with exogenous CK2 and GTPgammaS. The marked band is then excised from the gel and submitted for protein identification by mass spectrometry. Following this procedure, putative CK2 substrates identified by a mass spectrometry must be validated using an additional approach, such as by CK2 dependent phosphorylation in a standard in vitro kinase assay.

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Biochemistry

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

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

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

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

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 ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

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; ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

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 ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

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

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

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