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

这项工作得到韩国政府资助的国家研究基金会（MSIP）（第2015R1A2A1A10052663号和第2014M1A3A3A02034698号）的支持。

安全注意：本协议使用[γ- 32 P] -ATP评估AMPK的活性。磷32是放射性同位素，主要发射β辐射。由于β粒子的尺寸非常小，因此可以很容易地穿透衣服和皮肤。外部和内部暴露于β辐射可能对人体健康有害，包括引起皮肤灼伤和组织损伤。 <ol><li>执行需要使用[γ- 32 P] -ATP的所有步骤，使用适当的保护，如丙烯酸屏蔽。 </li><li>确保所有人员配备电子个人剂量计（EPD?...

<ol>
	<li>Hardie, D. G., Schaffer, B. E., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK:+An+energy-sensing+pathway+with+multiple+inputs+and+outputs.">AMPK: An energy-sensing pathway with multiple inputs and outputs.</a> Trends Cell Biol. 26, 190-201 (2016).</li><li>Gwinn, D. 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=AMPK+phosphorylation+of+raptor+mediates+a+metabolic+checkpoint.">AMPK phosphorylation of raptor mediates a metabolic checkpoint.</a> Mol Cell. 30, 214-226 (2008).</li><li>Huang, H. C., Nguyen, T., Pickett, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Nrf2+at+Ser-40+by+protein+kinase+C+regulates+antioxidant+response+element-mediated+transcription.">Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription.</a> J Biol Chem. 277, 42769-42774 (2002).</li><li>Niture, S. K., Jain, A. K., Jaiswal, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant-induced+modification+of+INrf2+cysteine+151+and+PKC-delta-mediated+phosphorylation+of+Nrf2+serine+40+are+both+required+for+stabilization+and+nuclear+translocation+of+Nrf2+and+increased+drug+resistance.">Antioxidant-induced modification of INrf2 cysteine 151 and PKC-delta-mediated phosphorylation of Nrf2 serine 40 are both required for stabilization and nuclear translocation of Nrf2 and increased drug resistance.</a> J Cell Sci. 122, 4452-4464 (2009).</li><li>Jain, A. K., Jaiswal, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GSK-3beta+acts+upstream+of+Fyn+kinase+in+regulation+of+nuclear+export+and+degradation+of+NF-E2+related+factor+2.">GSK-3beta acts upstream of Fyn kinase in regulation of nuclear export and degradation of NF-E2 related factor 2.</a> J Biol Chem. 282, 16502-16510 (2007).</li><li>Steen, H., Jebanathirajah, J. A., Rush, J., Morrice, N., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+analysis+by+mass+spectrometry:+myths,+facts,+and+the+consequences+for+qualitative+and+quantitative+measurements.">Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements.</a> Mol Cell Proteomics. 5, 172-181 (2006).</li><li>Dephoure, N., Gould, K. L., Gygi, S. P., Kellogg, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+analysis+of+phosphorylation+sites:+a+quick+guide+for+cell+biologists.">Mapping and analysis of phosphorylation sites: a quick guide for cell biologists.</a> Mol Biol Cell. 24, 535-542 (2013).</li><li>Joo, M. 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=AMPK+facilitates+nuclear+accumulation+of+Nrf2+by+phosphorylating+at+Serine+550.">AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at Serine 550.</a> Mol Cell Biol. 36, 1931-1942 (2016).</li><li>Sinclair, W. K., Holloway, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half-lives+of+some+radioactive+isotopes.">Half-lives of some radioactive isotopes.</a> Nature. 167 (4244), 365 (1951).</li><li>Chang, B. Y., Chiang, M., Cartwright, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+Src+and+RACK1+is+enhanced+by+activation+of+protein+kinase+C+and+tyrosine+phosphorylation+of+RACK1.">The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1.</a> J Biol Chem. 276, 20346-20356 (2001).</li><li>Smith, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RACK1+interacts+with+filamin-A+to+regulate+plasma+membrane+levels+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator.</a> Am J Physiol Cell Physiol. 305 (1), C111-C120 (2013).</li></ol>

作为评估由AMPK介导的预测的磷酸化位点的真实性的简单和方便的方法，在此我们描述了体外激酶测定法，其可用于使用竞争性肽发现特定磷酸化位点，并使用位点特异性突变体。从体外竞争性AMPK活性测定获得的代表性数据与使用位点定向突变蛋白的测定结果匹配，表明肽竞争测定是确定磷酸化位点的有用工具。该方法也用于鉴定由PKCδ磷酸化的特异性位点的研究，其在丝氨酸40 <sup c...

特定残基处的蛋白磷酸化在广泛的细胞过程中起着重要的作用。因此，了解信号网络需要鉴定特异性磷酸化位点。此外，磷酸化位点决定了对蛋白质功能的影响，因为蛋白质内的各个结构域具有不同的结构和功能。核因子红因子2相关因子2（Nrf2）的活性是一种主要的抗氧化转录因子，通过不同位点的磷酸化双向调节。我们的研究集中在催化Nrf2磷酸化的激酶。 Nrf2对氧化性攻击的应激反应迅速发生，主要通过丝氨酸40的磷酸化并由蛋白激酶C（PKC）-δ介导，其激活Nrf2,3,4。相反，Fyn催化Nrf2在酪氨酸5处的抑制性磷酸化68用于活动的紧密控制5 。 用于发现磷酸化位点的最常见方法是液相色谱/质谱（LC / MS）分析。可以通过这种方法生成快速高灵敏度的磷酸化位点作图数据;然而，它有几个技术限制，通常产生假阴性信号。 LC / MS分析中频繁发生序列覆盖不良。为了鉴定磷酸化位点，需要关于蛋白质最大氨基酸覆盖度的信息6 。在消化步骤期间与几种蛋白酶的目的蛋白质的蛋白水解可能有助于改善序列覆盖。识别磷酸化残基的另一个障碍是丝氨酸和苏氨酸磷酸化肽6,7经常观察到的磷酸的容易损失。不规则磷酸酯部分通常是在碎裂过程中从磷酸肽释放7 。寻找磷酸化位点时的第二个选择是使用肽微阵列法。可以使用含有衍生自感兴趣的蛋白质的肽片段的微阵列芯片筛选激酶靶位点。然而，由于微阵列芯片的生产和检测的设备要求，肽微阵列方法被认为是耗时且昂贵的。 为了克服这些挑战， 体外寡肽竞争激酶测定可用于具有已知识别基序的蛋白激酶。如果确定了激酶的共识识基序，则可以预测候选底物的推定的磷酸化位点，并且可以验证位点的真实性。这个程序最有说服力的方法是显示在其中是preicte的突变蛋白中磷酸化的消除d残基被不可磷酸化的氨基酸（ 即丝氨酸或苏氨酸转化为丙氨酸，酪氨酸至苯丙氨酸）取代。然而，突变蛋白的生产和分离是耗时的。作为研究初期的替代方案，竞争性肽激酶测定是直接和方便的。在这里，我们描述了体外肽竞争测定和磷酸化位点验证的方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>15630</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</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>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4</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>[&#947;-32P]-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</td>
			<td>Daejung Chemicals &#38; Metals, Siheung, South Korea</td>
			<td>5558-4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</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

特定位点的蛋白质磷酸化决定了其与其他分子的构象和相互作用。因此，蛋白磷酸化影响细胞的生物功能和特征。目前，发现磷酸化位点最常见的方法是通过液相色谱/质谱（LC / MS）分析，快速而灵敏的方法。然而，相对不稳定的磷酸酯部分通常在分裂步骤期间从磷酸肽释放，其通常产生假阴性信号。在这种情况下，使用定点突变体的传统的体外激酶测定将更准确，但是该方法费时费力。因此，使用肽竞争的替代方法可能是有利的。已经建立了5&#39;腺苷单磷酸激活蛋白激酶（AMPK）的共识识基序1 ，并使用位置扫描肽库文库验证ay 2 。因此，可以通过肽竞争测定来预测和证实用于新基质的AMPK磷酸化位点。在本报告中，我们通过说明AMPK介导的核因子红系2相关因子2（Nrf2）磷酸化来描述体外寡肽竞争激酶测定的详细步骤和程序。为了认证磷酸化位点，我们使用位点特异性突变体进行了连续的体外激酶测定。总体上，肽竞争测定提供了筛选多个潜在磷酸化位点并鉴定磷酸化位点突变体验证位点的方法。

图1和图2显示了先前报道的文献8中重复实验的结果。合成了三种模拟推定的AMPK靶位点（包括Ser153的＃1,148-157;包含Ser335的＃2，330-339;和包含Ser558的＃3，553-562）的10个残基寡肽，并将其用作在体外激酶测定。在寡肽＃3的存在下，AMPK对Nrf2的磷酸化作用大大降低（ 图1 ）。接下来，进...

Watch this Scientific Journal Video about Oligopeptide Competition Assay for Phosphorylation Site Determination at JoVE.com

Oligopeptide Competition Assay for Phosphorylation Site Determination

肽竞争测定广泛用于各种分子和免疫学实验。本文描述了体外寡肽竞争激酶测定的详细方法和相关的验证程序，其可用于发现特定的磷酸化位点。

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