Name: oligopeptide competition assay for phosphorylation site determination
Uploaded: 2017-05-18T14:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Oligopeptide Competition Assay for Phosphorylation Site Determination at JoVE.com

This work was supported by the National Research Foundation of Korea grant funded by the Korean government (MSIP) (No. 2015R1A2A1A10052663 and No. 2014M1A3A3A02034698).

1. Safety
CAUTION: This protocol uses [&#947;-32P]-ATP to assess the activity of AMPK. Phosphorus-32 is a radioactive isotope, largely emitting beta radiation. Since the size of a beta particle is extremely small, it can easily penetrate clothing and skin. Both external and internal exposure to beta radiation may be harmful to human health, including by causing skin burns and tissue damage.
<ol>
	<li>Carry out all the steps that require the use of [&#947;-32P]-ATP using proper protection, such as acrylic shielding.</li>
	<li>Ensure that all personnel are equipped with electronic personal dosimeters (EPD) to monitor the extent of exposure to harmful radiation during all procedures.</li>
	<li>Handle open-source radiation only after appropriate training and regulatory approval. 
	NOTE: Here, all experiments were conducted after the completion of training on open-source radiation use at the Seoul National University and the approval of the Korean government (nssc.go.kr/nssc/en).</li>
	<li>Dispose of radioactive waste per local regulations. 
	NOTE: Here, directives from the Institute of Environmental Protection &#38; Safety of the Seoul National University were followed. The followings are a list of regulators in the United States and European countries: The United States of America, United States Nuclear Regulatory Commission (http://www.nrc.gov/); United Kingdom, Health and Safety Executive (HSE) (http://www.hse.gov.uk); France, Autorit&#233; de s&#251;ret&#233; nucl&#233;aire (https://www.asn.fr/); and Germany, Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMU) (http://www.bmu.de).</li>
</ol>
2. In Vitro Competitive Kinase Assay
<ol>
	<li>Construction of Competitive Peptides
	<ol>
		<li>Determine the consensus motif phosphorylated by AMPK. 
		NOTE: AMPK recognizes the consensus motif, &#934;-[X,&#946;]-X-X-Ser/Thr-X-X-X-&#934;. In the consensus sequence, &#934; represents hydrophobic amino acids (i.e., valine [V], leucine [L], methionine [M], and isoleucine [I]) and &#946; represents basic amino acids (i.e., lysine [K], arginine [R], and histidine [H])1.</li>
		<li>Select serine or threonine residues from target sequences according to the above consensus motif. Use three putative sites of human Nrf2 (#1, 148-157 comprising Ser153; #2, 330-339 comprising Ser335; and #3, 553-562 comprising Ser558)8.</li>
		<li>Commercially synthesize 10-residue peptides that mimic the putative sites. Obtain the synthesized product as a lyophilized powder with a purity greater than 98% for each peptide.</li>
		<li>Dissolve the peptides using a kinase buffer to achieve a concentration of 71.667 g/L. If a peptide is not soluble in a kinase buffer, dissolve it in 20% DMSO. If it remains insoluble, a peptide mimicking the putative site may be extended to increase solubility.</li>
	</ol>
	</li>
</ol>
<ol start="2">
	<li>In Vitro Competitive AMPK Kinase Assay
	<ol>
		<li>Prepare 5&#215; kinase buffer as follows: 100 mM HEPES, pH 7.4; 25 mM MgCl2; 5 mM EGTA; 5 mM DTT; 125 mM &#946;-glycerophosphate (serine/threonine phosphatase inhibitor); 5 mM Na3VO4 (tyrosine phosphatase inhibitor); 0.5% (v/v) Protease Inhibitor Cocktail Set III, 1 mM cold ATP; and 500 &#181;M AMP. 
		NOTE: AMP is an essential component of the kinase buffer to test AMPK activity. The buffer can be used to measure the activity of both serine/threonine kinases and tyrosine kinases.</li>
		<li>Thaw the following recombinant protein and synthetic peptides on ice: AMPK; Nrf2; and oligopeptides #1, #2, and #3.</li>
		<li>Prepare a reaction buffer on ice: 0.15 &#181;g of AMPK, 0.4 &#181;g of Nrf2 (~4 pmol), 0.43 mg of oligopeptide (~300 nmol), and 6 &#181;L of 5&#215; kinase buffer. Adjust the final volume to 30 &#181;L with sterile distilled water (DW). 
		NOTE: The addition of [&#947;-32P]-ATP after preparing the reaction buffer may reduce the radioactive signal due to kinase autophosphorylation.</li>
	</ol>
	</li>
	<li>Running the Reactants 
	NOTE: All the processes below must be carried out under protection with an acrylic shield, rack, or block, as well as appropriate personal protective equipment.
	<ol>
		<li>Set heating blocks to 30 &#176;C before starting the experiment.</li>
		<li>Add 1 &#181;L of [&#947;-32P]-ATP (1 &#181;Ci) to the reaction tubes on ice. Mix the reaction buffer by pipetting up and down several times. 
		NOTE: The radioactivity of 32P has a short half-life of approximately 14 days9. Adjust the volume by considering the half-life (e.g., after one month from the production of [&#947;-32P]-ATP, add 4 &#181;L of [&#947;-32P]-ATP to make 1 &#181;Ci).</li>
		<li>Incubate the sample in the heating block at 30 &#176;C for 15-30 min. During this step, prepare 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Adjust the percentage of the gel according to the size of the target protein.</li>
		<li>Stop the kinase reaction by adding 3 &#181;L of 10&#215; SDS sample buffer (10% (v/v) glycerol, 20% (w/v) SDS, 15.42% (w/v) dithiothreitol, 90% (v/v) 0.5 M Tris-HCl (pH 6.8), and 0.02% (w/v) bromophenol blue). Mix the reaction buffer by pipetting up and down several times.</li>
		<li>Run the samples in a 7.5% SDS-PAGE gel at 70 V for 20 min and continuously at 140 V for 1 h. 
		NOTE: The bromophenol blue line at the end of the gel contains extremely high radioactive signals. It is recommended to remove the gel below the blue line before proceeding to the next step to reduce background signals.</li>
		<li>After SDS-PAGE, gently remove the gel from the caster. Place the gel in a glass tube for the next step.</li>
		<li>Fix the gel with fixation buffer for 20 min (50% methanol and 10% glacial acetic acid).</li>
		<li>Gently move the gel onto filter paper and cover it with a transparent wrapper. Note that the gel may be easily torn at this step. Dry the gel with a vacuum gel dryer at 80 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Visualization
	<ol>
		<li>Expose the gel to either a phosphor screen or an X-ray film overnight (usually for approximately 16 h). 
		NOTE: A phosphor screen can be reused after exposure to the extra-bright light. When an X-ray film is used, expose the gel over two days. X-ray films have lower sensitivities than phosphor screens.</li>
		<li>Scan the phosphor screen with a phosphor imager. Export the image as a high-resolution TIFF or bitmap file.</li>
		<li>After visualization, move the gel to a glass or plastic container for Coomassie brilliant blue (CBB) staining.</li>
		<li>Wash the gel with DW. Discard the DW and stain the gel with the staining reagent for 1 h.</li>
		<li>Discard the reagent and destain the gel with DW for 1 h or overnight. Place the gel between transparent plastic films (or the equivalent) to scan with an office scanner.</li>
	</ol>
	</li>
</ol>
3. In Vitro Kinase Assay Using a Site-specific Mutant
<ol>
	<li>Site-Directed Mutagenesis of pGEX-Nrf2
	<ol>
		<li>Mutagenic Primer Design 
		NOTE: Designing proper mutagenic primers is critical for the successful outcome of mutagenesis. The following are some considerations for designing mutagenic primers. Create a primer consisting of 25 to 45 bases, with the desired mutation in the middle of the primer. The primer should have 40-60% GC content and terminate with one or more G or C bases. Ensure that the Tm of the primer is equal to 78 &#176;C or greater according to the formula [Tm= 81.5 + 0.41(% GC) - (675/length of bases) - % mismatch]. 
		NOTE: Purify the primer because its purity is important for mutation efficiency. There are several websites for designing mutagenic primers.
		<ol>
			<li>Open the primer design program for &#34;designing primers for site-directed mutagenesis&#34; (http://www.genomics.agilent.com/primerDesignProgram.jsp).</li>
			<li>Paste human Nrf2 DNA sequence (Access No. NM_006164.4) and select the &#34;upload translated&#34; button.</li>
			<li>Select the serine 558 residue from the translated amino acid sequence to be changed to alanine. Using the mutagenic primer, substitute the codon for serine from agc to gcc, for alanine.</li>
			<li>Consider primer length, Tm, and the GC content. 
			NOTE: The mutagenic primer is finally designed as 5&#39;-ctactgaaaaaacaactc gccaccttatatctcgaag-3&#39;.</li>
		</ol>
		</li>
		<li>Reaction for Mutant Strand Synthesis using Polymerase Chain Reaction (PCR)
		<ol>
			<li>Prepare the PCR reaction mixture on ice: 10&#215; reaction buffer, 50 ng of pGEX-GST-Nrf2, 125 ng of forward primer, 125 ng of reverse primer, 1 &#181;L of dNTP mix (10 mM each), 2.5 U Pfu DNA polymerase, and sterile DW to 50 &#181;L.</li>
			<li>Run the PCR for pGEX-GST-Nrf2-mutant strand synthesis with the following cycling program: 1 cycle at 95 &#176;C for 30 s; 18 cycles at 95 &#176;C for 30 s, at 55 &#176;C for 1 min, and at 68 &#176;C for 90 s; and 1 cycle at 68 &#176;C for 5 min. 
			NOTE: Depending on the plasmid length, the cycling parameters may be changed.</li>
			<li>Place it on ice for 2 min to cool the reactants for the next step. Alternatively, store them at 4 &#176;C overnight.</li>
		</ol>
		</li>
		<li>Dpn I Digestion and the Selection of a Mutant Plasmid
		<ol>
			<li>Add 1 &#181;L of Dpn I restriction enzyme (10 U/&#181;L) to each of the reactants after cooling. Mix thoroughly and gently by pipetting and centrifuge for 1 min.</li>
			<li>Incubate at 37 &#176;C for 2 h to digest parental pGEX-GST-Nrf2 double-stranded DNA.</li>
			<li>Gently thaw DH5&#160;&#945; supercompetent cells on ice.</li>
			<li>Add 50 &#181;L of the reactant to 150 &#181;L of pre-chilled DH5 &#945; supercompetent cells and incubate on ice for at least 30 min.</li>
			<li>Transfer the tubes to a heating block preheated to 42 &#176;C and leave them for 90 s. Place them on ice for 2 min.</li>
			<li>Add pre-warmed lysogeny broth (LB) to the transformed DH5&#160;&#945; cells and incubate at 37 &#176;C for 1 h with shaking at 180 rpm.</li>
			<li>Spread the transformed cells on LB-ampicillin agar plates and incubate overnight at 37 &#176;C. 
			NOTE: Ampicillin is used for selection since the plasmid GEX-GST-Nrf2 has the ampicillin resistance marker. Use appropriate antibiotics, depending on the resistance marker of the mutagenized plasmid.</li>
			<li>Pick a single colony from the plate, transfer it to 5 mL of LB-ampicillin, and incubate overnight it at 37 &#176;C with shaking at 180 rpm. For verification of the mutagenic sequence, evaluate at least three colonies.</li>
			<li>Extract DNA using a DNA miniprep kit for DNA sequence analysis; follow the manufacturer&#39;s protocol.</li>
			<li>Verify the mutagenic DNA sequences using an automatic DNA sequence analyzer according to the manufacturer&#39;s protocol. Use at least 200 ng of the DNA construct for the verification of mutant sequences.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Purification of Recombinant GST-Nrf2 Protein
	<ol>
		<li>Expression of Mutagenic GST-Nrf2 Fusion Protein in Escherichia coli
		<ol>
			<li>Transform the mutagenic pGEX-GST-Nrf2 plasmid into BL21 cells. Inoculate a single colony in 25 mL of LB broth containing ampicillin (100 &#181;g/mL) and incubate at 37 &#176;C on a rotator overnight.</li>
			<li>Inoculate the cultured cells in 100 mL of LB broth containing ampicillin (100 &#181;g/mL) at a 1:100 dilution ratio.</li>
			<li>Incubate at 37 &#176;C on a rotator until the absorbance at 600 nm reaches around 0.4-0.8 (approximately 4 h).</li>
			<li>Add 100 &#181;L of 1 M IPTG to 100 mL of cultured cells to prepare a final concentration of 1 mM IPTG.</li>
			<li>Incubate the cells at 30 &#176;C on a rotator for 6 h or overnight for protein expression.</li>
			<li>Centrifuge the cells at 5,000 x g for 20 min and resuspend the cell pellets with 1 mL of bacterial lysis buffer (25 mM HEPES, 5 mM EDTA, 2 mM DTT, 0.1% CHAPS, 1 &#181;g/mL pepstatin, 0.5 &#181;g/mL leupeptin, and 1 mM PMSF) on ice.</li>
			<li>Lyse the cell pellets using an ultrasonic homogenizer with 2-s intervals for 2 min at 50% of the maximum power output.</li>
			<li>Centrifuge the cells at 12,000&#160;&#215; g for 15 min at 4 &#176;C and collect the supernatant.</li>
		</ol>
		</li>
		<li>Purification of Recombinant GST-Nrf2 Protein
		<ol>
			<li>Prepare 200 &#181;L of 50% (v/v) glutathione-agarose beads and wash with 1 mL PBS (pH 7.3; 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4). Collect the beads by centrifugation at 500&#160;&#215; g for 1 min. Repeat the washing step three times.</li>
			<li>Add the cell lysate to the prepared 200 &#181;L of 50% (v/v) glutathione-agarose beads and incubate at 4 &#176;C on an end-over-end rotator for 2 h.</li>
			<li>Collect the glutathione-agarose beads combined with GST-Nrf2 protein by centrifugation at 500 x g for 1 min. Remove the supernatant using a 1 mL syringe with a 31 G needle.</li>
			<li>Wash the beads with 1 mL of PBS (pH 7.3), as in step 1.</li>
			<li>Add 500 &#181;L of elution buffer (50 mM Tris-HCl and 10 mM reduced glutathione, pH 8.0) to the glutathione-agarose beads combined with GST-Nrf2. Incubate at 4 &#176;C on an end-over-end rotator for 30 min.</li>
			<li>Centrifuge at 500 x g for 1 min and collect the supernatant.</li>
			<li>Again add 300 &#181;L of elution buffer to the beads and repeat steps 3.2.2.5-3.2.2.6 twice.</li>
		</ol>
		</li>
		<li>Confirmation of the Purified GST-Nrf2 Protein
		<ol>
			<li>Prepare 5 &#181;L of the sample for visualization of protein quantity with 0.5 &#181;g of standard protein BSA.</li>
			<li>Run the samples in a 7.5% SDS-PAGE gel at 70 V for 20 min and then at 140 V for 60 min.</li>
			<li>Stain the gel with 0.1% CBB staining solution (0.1 g of CBB R250, 40 mL of 99% methanol, 10 mL of glacial acetic acid, and 50 mL of distillated H2O) for 2 h and destain (30 mL of methanol, 10 mL of glacial acetic acid, and 60 mL of distilled H2O) until the bands are clearly shown.</li>
			<li>Place the destained gel on filter paper and cover with a transparent wrapper. Dry the gel with a vacuum gel dryer at 80 &#176;C for 1 h.</li>
			<li>Determine the quantity of mutant GST-Nrf2 protein with densitometry for the in vitro AMPK kinase assay.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>In Vitro AMPK Activity Assay with a Site-Specific Mutant 
	NOTE: The in vitro AMPK kinase assay is performed according to steps 2.2-2.4, using 0.4 &#181;g of purified wildtype GST-Nrf2 or the GST-Nrf2-S558A mutant without peptide inhibitors. Follow all the radiation safety guidelines described in step 1.
	<ol>
		<li>Prepare a reaction buffer on ice with 0.15 &#181;g of AMPK, 0.4 &#181;g of mutant or wildtype GST-Nrf2, and 6 &#181;L of 5 kinase buffer. Fill up to 30 &#181;L with sterile DW.</li>
		<li>Add 1 &#181;L of [&#947;-32P]-ATP (1 &#181;Ci/&#181;L) to the reaction tubes on ice. Mix the reaction buffer by pipetting up and down several times and centrifuge.</li>
		<li>Incubate the samples in a heating block at 30 &#176;C for 30 min.</li>
		<li>Stop the kinase reaction by adding 3 &#181;L of 10&#215; SDS sample buffer.</li>
		<li>Run in a 7.5% SDS-PAGE gel at 70 V for 20 min and then at 140 V for 1 h.</li>
		<li>Fix the gel with the fixation buffer for 20 min, move it onto the filter paper, and cover the gel with a transparent wrapper.</li>
		<li>Dry the gel with the vacuum gel dryer at 80 &#176;C for 1 h and then expose it overnight to either a phosphor screen or an X-ray film.</li>
		<li>Scan the phosphor screen with a phosphor imager and move the gel to a glass tube for CBB staining after visualization according to step 2.4.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

As a simple and convenient way to assess the authenticity of the predicted phosphorylation sites mediated by AMPK, here we describe an in vitro kinase assay that can be used to discover a specific phosphorylation site using competitive peptides and to verify it using a site-specific mutant. The representative data obtained from the in vitro competitive AMPK activity assay matched the results from an assay using a site-directed mutant protein, indicating that the peptide competition assay is a useful too...

Protein phosphorylation at a specific residue plays a significant role in a wide range of cellular processes. Thus, an understanding of signaling networks requires the identification of specific phosphorylation sites. In addition, the phosphorylation site determines the effect on protein function because individual domains within a protein possess different structures and functions. The activity of nuclear factor erythroid 2-related factor 2 (Nrf2), a key antioxidant transcription factor, is bi-directionally regulated through phosphorylation at different sites. Our studies have focused on the kinases that catalyze the phosphorylation of Nrf2. The stress response of Nrf2 against oxidative challenge occurs rapidly, mainly through phosphorylation at serine 40 and mediated by protein kinase C (PKC)-&#948;, which activates Nrf23,4. Conversely, Fyn catalyzes the inhibitory phosphorylation of Nrf2 at tyrosine 568 for tight control of the activity5.
The most common method used to discover phosphorylation sites is liquid chromatography/mass spectrometry (LC/MS) analysis. Rapid and highly sensitive phosphorylation site mapping data can be generated this way; however, it has several technical limitations, often generating false-negative signals. Poor sequence coverage frequently occurs in LC/MS analysis. To identify phosphorylation sites, information on the maximal amino acid coverage of a protein is required6. Proteolysis of the protein of interest with several proteases during the digestion step may be of help in improving sequence coverage. Another obstacle for the identification of phosphorylation residues is the facile loss of phosphoric acid that is frequently observed for serine- and threonine-phosphorylated peptides6,7. Labile phosphate moieties are often released from phosphopeptides during the fragmentation process7. The second option when searching for phosphorylation sites is using a peptide microarray method. It is possible to screen for the kinase target sites using a microarray chip containing peptide fragments derived from a protein of interest. However, due to the equipment requirement for the production and detection of a microarray chip, the peptide microarray method is considered time-consuming and expensive.
To overcome these challenges, an in vitro oligopeptide-competing kinase assay can be used for a protein kinase with known recognition motifs. If the consensus recognition motif of a kinase is established, putative phosphorylation sites of a candidate substrate can be predicted, and the authenticity of the sites can be validated. The most convincing method for this procedure is to show the abrogation of phosphorylation in a mutant protein in which the predicted residue is substituted with a non-phosphorylatable amino acid (i.e., serine or threonine to alanine; tyrosine to phenylalanine). However, the production and isolation of mutant proteins is time-consuming. As an alternative at the initial stage of research, the competitive peptide kinase assay is straightforward and convenient. Here, we describe a protocol for an in vitro peptide competition assay and for the validation of the phosphorylation site.

<table><tbody><tr><td>HEPES</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>15630</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louis, MO</td><td>208337</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich, St. Louis, MO</td><td>E3889</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich, St. Louis, MO</td><td>D9779</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma-Aldrich, St. Louis, MO</td><td>G9422</td><td></td></tr><tr><td>Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louis, MO</td><td>450243</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Calbiochem, Nottingham, UK</td><td>539134</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A2383</td><td></td></tr><tr><td>AMP</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A1752</td><td></td></tr><tr><td>AMPK</td><td>Upstate Biotechnology, Lake Placid, NY</td><td>14-840</td><td></td></tr><tr><td>Nrf2 (WT)</td><td>Abnova, Taipei City, Taiwan</td><td>H00004780-P01</td><td></td></tr><tr><td>[&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P]-ATP</td><td>PerkinElmer Life and Analytical Sciences, Waltham, MA</td><td>NEG502A</td><td></td></tr><tr><td>EZblue staining reagent</td><td>Sigma-Aldrich, St. Louis, MO</td><td>G1041</td><td></td></tr><tr><td>Pfu turbo DNA polymerase</td><td>Agilent Technologies, Santa Clara, CA</td><td>600250</td><td></td></tr><tr><td>dNTP mix</td><td>Agilent Technologies, Santa Clara, CA</td><td>200415-51</td><td>Avoid multiple thaw and freezing cycle</td></tr><tr><td>DpnI</td><td>New England Biolabs, Ipswich, MA</td><td>R0176S</td><td></td></tr><tr><td>LB broth</td><td>Duchefa Biochemie BV, Haarlem, Netherlands</td><td>L1704</td><td></td></tr><tr><td>Ampicillin</td><td>Affymetrix, Santa Clara, CA</td><td>11259</td><td></td></tr><tr><td>Agarose LE</td><td>iNtRON Biotechnology, Sungnam, South Korea</td><td>32034</td><td></td></tr><tr><td>HiYield Plus Gel/PCR DNA Mini Kit</td><td>Real Biotech Corporation, Taipei, Taiwan</td><td>QDF100</td><td></td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>161-0400</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Bovogen Biologicals, Victoria, Australia</td><td>BSA100</td><td></td></tr><tr><td>Glutathione Sepharose 4B</td><td>GE Healthcare, Marlborough, MA</td><td>17-0756-01</td><td></td></tr><tr><td>Acetic Acid glacial</td><td>Duksan pure chemicals, Ansan, South Korea</td><td></td><td></td></tr><tr><td>Methyl alcohol&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;</td><td>Daejung Chemicals &amp;amp; Metals, Siheung, South Korea</td><td>5558-4410</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Typhoon FLA 7000</td><td>GE Healthcare, Marlborough, MA</td><td>28-9558-09</td><td></td></tr><tr><td>SDS-PAGE kit</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>1658001FC</td><td></td></tr><tr><td>Vacuum pump</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>165-178</td><td></td></tr><tr><td>Gel dryer</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>165-1746</td><td></td></tr><tr><td>Dancing shaker</td><td>FINEPCR, Seoul, Korea</td><td>CR300</td><td>The machine is needed for washing step</td></tr><tr><td>PCR machine</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>T100</td><td></td></tr><tr><td>Incubator/shakers</td><td>N-BIOTEK, GyeongGi-Do, Korea</td><td>NB-205L</td><td></td></tr><tr><td>Microcentrifuges</td><td>LABOGENE, Seoul, Korea</td><td>1730R</td><td></td></tr><tr><td>Chromatography columns</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>732-1010</td><td></td></tr></tbody></table>

oligopeptide competition assay for phosphorylation site determination

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

Figure 1 and Figure 2 demonstrate the outcomes from repeated experiments in the previously reported paper8. Three different 10-residue oligopeptides mimicking the putative AMPK target sites (#1, 148-157 comprising Ser153; #2, 330-339 comprising Ser335; and #3, 553-562 comprising Ser558) were synthesized and used as competitors in the in vitro kinase assay. The phosphorylation of Nrf2 by AMPK was g...

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Oligopeptide Competition Assay for Phosphorylation Site Determination

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

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

oligopeptide-competition-assay-for-phosphorylation-site-determination

Research

JoVE Journal

Biochemistry

8.4K Views.  Seoul National University. Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Video: Oligopeptide Competition Assay for Phosphorylation Site Determination

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; ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

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

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

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

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

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

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

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

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 ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Immunology and Infection

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Biology

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

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Assaying Protein Kinase Activity with Radiolabeled ATP

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

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

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

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Identification of Post-translational Modifications of Plant Protein Complexes

Identification of Kinase-substrate Pairs Using High Throughput Screening

Comparing the Affinity of GTPase-binding Proteins using Competition Assays