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），以监测所有程序中暴露于有害辐射的程度。 </li><li>只有经过适当的培训和监管批准才能处理开源辐射。 注:在这里，所有的实验都是在首尔国立大学完成开源辐射使用培训后进行的呃和韩国政府的批准（nssc.go.kr/nssc/en）。 </li><li>根据当地法规处理放射性废物。 注:遵循首尔国立大学环境保护与安全研究所的指示。以下是美国和欧洲国家的监管机构名单:美利坚合众国，美国核管理委员会（http://www.nrc.gov/）;英国健康与安全执行（HSE）（http://www.hse.gov.uk）;法国，自由诺基亚（https://www.asn.fr/）;和德国，联邦环境，自然保护，建筑和核安全部（BMU）（http://www.bmu.de）。 </li></ol> 2. 体外竞争激酶测定<ol><li> 竞争肽的构建 <ol><li>确定由AMPK磷酸化的共有基序。 注意:AMPK识别共识基序，＃934; - [X，β] -XX-丝氨酸/苏氨酸-XXX-Φ。在共识序列中，Φ表示疏水氨基酸（ 即缬氨酸[V]，亮氨酸[L]，甲硫氨酸[M]和异亮氨酸[I]），β代表碱性氨基酸（ 即赖氨酸[K]，精氨酸[ R]和组氨酸[H]） 1 。 </li><li>根据上述共识基序从目标序列中选择丝氨酸或苏氨酸残基。使用人Nrf2的三个假定位点（＃1，包括Ser153的148-157;包含Ser335的＃2，330-339;和包含Ser558的＃3，553-562） 8 。 </li><li>商业合成模拟推定位点的10位残基肽。获得合成的产物作为每种肽的纯度大于98％的冻干粉末。 </li><li>使用激酶缓冲液溶解肽以达到71.667g / L的浓度。如果肽不溶于激酶缓冲液，则将其溶于20％DMSO。如果它是不溶性的，则模仿放置的肽可能延长现场可增加溶解度。 </li></ol></li></ol><ol start="2"><li> 体外竞争性AMPK激酶测定 <ol><li>如下制备5×激酶缓冲液:100mM HEPES，pH 7.4; 25mM MgCl 2 ; 5mM EGTA; 5mM DTT; 125mMβ-甘油磷酸酯（丝氨酸/苏氨酸磷酸酶抑制剂）; 5mM Na 3 VO 4 （酪氨酸磷酸酶抑制剂）; 0.5％（v / v）蛋白酶抑制剂鸡尾酒套装III，1mM冷ATP;和500μMAMP。 注意:AMP是测试AMPK活性的激酶缓冲液的重要组成部分。缓冲液可用于测量丝氨酸/苏氨酸激酶和酪氨酸激酶的活性。 </li><li>在冰上解冻以下重组蛋白和合成肽:AMPK; Nrf2的;和寡肽＃1，＃2和＃3。 </li><li>在冰上制备反应缓冲液:0.15μgAMPK，0.4μgNrf2（〜4pmol），0.43mg寡肽（〜300nmol）和6μL5×激酶缓冲液。一个用无菌蒸馏水（DW）将最终体积调节至30μL。 注意:在制备反应缓冲液后添加[γ- 32 P] -ATP可能会降低由于激酶自磷酸化引起的放射性信号。 </li></ol></li><li> 运行反应物 注意:以下所有过程必须使用丙烯酸屏蔽，机架或块，以及适当的个人防护设备进行保护。 <ol><li>在开始实验之前将加热块设置为30°C。 </li><li>在冰上向反应管中加入1μL[γ- 32 P] -ATP（1μCi）。通过上下移动多次混合反应缓冲液。 注意: 32 P的放射性半衰期约为14天9 。通过考虑半衰期来调节体积（ 例如，从[γ- 32 P] -ATP生产一个月后，加入4μL[γ- 32 P]-ATP使1μCi）。 </li><li>将加热块中的样品在30℃下孵育15-30分钟。在此步骤中，制备7.5％十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶。根据目标蛋白的大小调整凝胶的百分比。 </li><li>通过加入3μL10×SDS样品缓冲液（10％（v / v）甘油，20％（w / v）SDS，15.42％（w / v）二硫苏糖醇，90％（v / v） M Tris-HCl（pH6.8）和0.02％（w / v）溴酚蓝）。通过上下移动多次混合反应缓冲液。 </li><li>将样品在7.5％SDS-PAGE凝胶中在70V下运行20分钟，并在140V持续1小时。 注意:凝胶末端的溴酚蓝线含有极高的放射性信号。建议在蓝色线下方移除凝胶，然后再进行下一步以减少背景信号。 </li><li> SDS-PAGE后，从施法者轻轻取出凝胶。将凝胶放在玻璃杯中ube为下一步。 </li><li>用固定缓冲液固定凝胶20分钟（50％甲醇和10％冰醋酸）。 </li><li>将凝胶轻轻移动到滤纸上，并用透明包装纸盖上。注意，在该步骤中，凝胶可能容易撕裂。用真空凝胶干燥器在80℃下干燥凝胶1小时。 </li></ol></li><li> 可视化 <ol><li>将凝胶暴露于荧光屏或X射线胶片过夜（通常约16小时）。 注意:荧光屏在暴露于超亮的光线之后可以重复使用。当使用X光胶片时，将凝胶暴露两天。 X射线胶片具有比荧光屏更低的灵敏度。 </li><li>用荧光成像仪扫描荧光屏。将图像导出为高分辨率TIFF或位图文件。 </li><li>在可视化后，将凝胶移动到玻璃或塑料容器，以进行考马斯亮蓝（CBB）染色。 </li><li>用DW洗涤凝胶。丢弃DW并染色e用染色试剂凝胶1小时。 </li><li>弃去试剂并用DW将凝胶脱胶1小时或过夜。将凝胶放在透明塑料薄膜（或等同物）之间，用办公扫描仪进行扫描。 </li></ol></li></ol> 3.使用位点特异性突变体进行体外激酶测定<ol><li> pGEX-Nrf2的定点诱变 <ol><li>诱变引物设计 注意:设计适当的诱变引物对于诱变的成功结果至关重要。以下是设计诱变引物的一些考虑。创建一个由25到45个碱基组成的引物，在引物中间有所需的突变。引物应具有40-60％的GC含量，并终止于一个或多个G或C碱基。根据公式[T m = 81.5 + 0.41（％GC） - （675 /碱基长度） - ％不匹配），确保引物的T m等于78℃或更高。 注意:净化引物，因为其纯度对突变效率很重要。有几个设计诱变引物的网站。 <ol><li>打开"定点诱变设计引物"的引物设计程序（http://www.genomics.agilent.com/primerDesignProgram.jsp）。 </li><li>粘贴人类Nrf2 DNA序列（访问号NM_006164.4）并选择"上传翻译"按钮。 </li><li>从翻译的氨基酸序列中选择丝氨酸558残基，以更改为丙氨酸。使用诱变引物，将密码子替换为来自agc的丝氨酸至gcc，用于丙氨酸。 </li><li>考虑引物长度，T m和GC含量。 注意:诱变引物最终设计为5&#39;-ctactgaaaaaacaactc gcc accttatatctcgaag-3&#39;。 </li></ol></li><li>使用聚合酶链反应（PCR）的突变链合成反应<ol><li>在冰上准备PCR反应混合物:10×反应缓冲液，50ngpGEX-GST-Nrf2，125ng正向引物，125ng反向引物，1μLdNTP混合物（每个10mM），2.5U Pfu DNA聚合酶和无菌DW至50μL。 </li><li>用以下循环程序运行pGEX-GST-Nrf2突变体链合成的PCR:95℃30秒的1个循环;在95℃30秒，55℃1分钟，68℃90秒循环18个循环;在68℃下1个循环5分钟。 注意:根据质粒长度，循环参数可能会改变。 </li><li>将其放在冰上2分钟，冷却反应物进行下一步骤。或者，将其储存在4°C过夜。 </li></ol></li><li> Dpn I消化和突变质粒的选择<ol><li>冷却后，向每个反应物中加入1μLDpn I限制酶（10U /μL）。通过移液彻底混合并离心1分钟。 </li><li>在37℃下孵育2小时以消化亲本pGEX-GST-Nrf2双双链DNA。 </li><li>在冰上轻轻融化DH5α超感受态细胞。 </li><li>将50μL反应物加入到150μL预冷的DH5α超级感受态细胞中，并在冰上孵育至少30 min。 </li><li>将管转移到预热至42℃的加热块，并将其放置90秒。将它们放在冰上2分钟。 </li><li>向转化的DH5α细胞中加入预热的溶菌酵母（LB），并在37℃下以180rpm摇动孵育1小时。 </li><li>将转化的细胞在LB-氨苄青霉素琼脂平板上铺展并在37℃下孵育过夜。 注意:氨苄青霉素用于选择，因为质粒GEX-GST-Nrf2具有氨苄青霉素抗性标记。使用适当的抗生素，这取决于诱变质粒的抗性标记。 </li><li>从板上挑取单个菌落，将其转移至5mL LB-氨苄青霉素中，并在37℃下以180rpm摇动孵育过夜。为了验证诱变序列，评估至少三个菌落。 </li><li>使用DNA微量制备试剂盒提取DNA进行DNA序列分析;遵循制造商的协议。 </li><li>使用自动DNA序列分析仪根据制造商的方案验证诱变DNA序列。使用至少200ng的DNA构建体来验证突变序列。 </li></ol></li></ol></li><li> 重组GST-Nrf2蛋白的纯化 <ol><li>诱变GST-Nrf2融合蛋白在大肠杆菌中的表达 <ol><li>将诱变型pGEX-GST-Nrf2质粒转化到BL21细胞中。接种在含有氨苄青霉素（100μg/ mL）的25mL LB肉汤中的单个菌落，并在37℃下在旋转器上孵育过夜。 </li><li>将培养的细胞接种在含有氨苄青霉素（100μg/ mL）的100mL含有1:100稀释比的LB肉汤中。 </li><li>在37°C的温度下旋转或直到600nm处的吸光度达到约0.4-0.8（约4小时）。 </li><li>向100 mL培养细胞中加入100μL1M IPTG，制备终浓度为1 mM IPTG。 </li><li>将细胞在30℃下在旋转器上孵育6小时或过夜以进行蛋白质表达。 </li><li>以5,000×g离心细胞20分钟，并用1mL细菌裂解缓冲液（25mM HEPES，5mM EDTA，2mM DTT，0.1％CHAPS，1μg/ mL胃酶抑素，0.5μg/ mL亮抑酶肽，和1mM PMSF）。 </li><li>使用超声波均化器以最大功率输出的50％，以2秒间隔裂解细胞沉淀2分钟。 </li><li>在4℃离心细胞12,000×g 15分钟并收集上清液。 </li></ol></li><li>重组GST-Nrf2蛋白的纯化<ol><li>准备200μL的50％（v / v）谷胱甘肽 - 琼脂糖珠，并用1mL PBS（pH 7.3; 140mM NaCl，2.7mM KCl，10mM Na 2 </sHPO 4和1.8mM KH 2 PO 4 ）。通过在500×g离心1分钟收集珠子。重复洗涤步骤三次。 </li><li>将细胞裂解物加入到制备的200μL的50％（v / v）谷胱甘肽 - 琼脂糖珠中，并在4℃下在终端旋转器上孵育2小时。 </li><li>通过在500×g离心1分钟收集与GST-Nrf2蛋白结合的谷胱甘肽 - 琼脂糖珠。用31 G针头的1 mL注射器取出上清液。 </li><li>用1mL PBS洗涤珠子（pH7.3），如步骤1。 </li><li>向与GST-Nrf2组合的谷胱甘肽 - 琼脂糖珠中加入500μL洗脱缓冲液（50mM Tris-HCl和10mM还原型谷胱甘肽，pH8.0）。在4℃下在终端旋转器上孵育30分钟。 </li><li>以500×g离心1分钟并收集上清液。 </li><li>再次加入洗脱缓冲液300μL，重复步骤3.2.2.5-3.2.2.6两次。 </li></ol></li><li>纯化的GST-Nrf2蛋白的确认<ol><li>准备5μL样品，用0.5μg标准蛋白质BSA观察蛋白质数量。 </li><li>将样品在7.5％SDS-PAGE凝胶中在70V下运行20分钟，然后在140V下运行60分钟。 </li><li>用0.1％CBB染色溶液（0.1g CBB R250,40mL99％甲醇，10mL乙酸乙酸和50mL蒸馏H 2 O）将凝胶染色2小时，脱色（​​30mL甲醇， 10mL冰醋酸和60mL蒸馏水H 2 O）直到清楚显示条带。 </li><li>将脱色凝胶放在滤纸上，盖上透明包装纸。用真空凝胶干燥器在80℃下干燥凝胶1小时。 </li><li>通过体外 AMPK激酶测定的光密度测定法确定突变体GST-Nrf2蛋白的数量。 </li></ol></li></ol></li><li> 体外 AMPK活性测定与位点特异性突变体 不E:根据步骤2.2-2.4 进行体外 AMPK激酶测定，使用0.4μg纯化的野生型GST-Nrf2或没有肽抑制剂的GST-Nrf2-S558A突变体。遵循步骤1中描述的所有辐射安全准则。 <ol><li>用0.15μgAMPK，0.4μg突变体或野生型GST-Nrf2和6μL5激酶缓冲液在冰上制备反应缓冲液。用无菌DW填充30μL。 </li><li>在冰上向反应管中加入1μL[γ- 32 P] -ATP（1μCi/μL）。通过上下移动多次混合反应缓冲液并离心。 </li><li>将样品在30℃加热块中孵育30分钟。 </li><li>加入3μL10×SDS样品缓冲液停止激酶反应。 </li><li>在7.5V的SDS-PAGE凝胶中在70V下运行20分钟，然后在140V下运行1小时。 </li><li>用固定缓冲液固定凝胶20分钟，将其移至滤纸上，并用透明膜覆盖凝胶包装</li><li>用真空凝胶干燥器在80℃下干燥凝胶1小时，然后将其过夜曝光至荧光屏或X射线胶片。 </li><li>用磷光体成像仪扫描荧光屏，并根据步骤2.4将凝胶移动到可视化后的玻璃管进行CBB染色。 </li></ol></li></ol>

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作者没有什么可以披露的。

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 3处磷酸化Nrf2。由于肽与蛋白质竞争性结合，所以该原理也可以应用于体外 GST下拉测定以鉴定结合基序10,11 。此外，这个肽tic方法也可用于鉴定其他翻译后修饰，例如赖氨酸残基处的乙酰化。 使用该方案，可能难以溶解一些疏水肽。如果肽不溶于激酶缓冲液，将其溶于20％DMSO可能是有帮助的。为了溶解极度疏水的肽，首先尝试将其溶解于100％DMSO中，然后用激酶缓冲液稀释溶液。超声处理也可能有助于溶解肽。如果保持不溶性，则应延长模拟推定位点的肽以增加溶解度。 本文介绍的方案描述了其中纯化的激酶和底物反应的" 体外系统"。在体外系统中，竞争性肽的浓度差异不会影响结果，除非浓度低于底物蛋白。 r从体外实验获得的结果包括作为通过人造接近和高浓度反应物产生的人为因素事件的固有可能性。为了消除这种前景，数据的解释应该伴随着基于细胞的实验的结果。使用肽获得假阳性结果的另一种可能是由于底物构象和/或激酶识别的改变。为了排除这种可能性，有必要用突变蛋白进行确认实验。 另一方面，也可以从该系统产生假阴性数据。许多激酶在细胞环境中充当蛋白质复合物，并且需要完整功能的辅因子。 AMPK是由AMPKα，AMPKβ和AMPKγ亚基构成的代表性蛋白激酶复合物，AMP与AMPKγ亚基的结合对激酶的激活是必需的。的确，AMPK没有在无AMP激酶缓冲液中磷酸化Nrf2（数据未显示）。因此，应当注意，AMP必须溶解在激酶反应缓冲液中用于成功的体外测定。 使用质谱法鉴定磷酸化位点是最流行的方法，具有几个技术限制，通常在碎裂过程中通过磷酸的容易损失产生假阴性信号7 。肽微阵列法可以成为第二个寻找磷酸化位点的选择。然而，这种方法被认为是耗时且昂贵的。作为筛选推定的磷酸化位点的替代方法，竞争性肽方法可以是一种方便和便宜的方式来了解推定的磷酸化位点的性质。

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><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

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，进行定点诱变测定以验证拟肽实验的结果。鉴于寡肽＃3对Nrf2的AMPK介导的磷酸化的抑制作用（ 图1 ），我们产生了S558A-Nrf2突变体。在野生型Nrf2和S之间比较磷酸化水平558A突变体使用体外 AMPK激酶测定。 Nrf2（Ser→Ala在558处）的单个氨基酸取代阻止AMPK磷酸化Nrf2，表明AMPK在Ser558处直接磷酸化Nrf2（ 图2 ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55708/55708fig1.jpg" /> 图1: 体外竞争性AMPK激酶测定。 在指定的寡肽（包括Ser153的＃1,148-157;包含Ser335的＃2，330-339;和包含Ser558的＃3，553-562）的存在下进行体外激酶测定。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55708/55708fig2.jpg" /> 图2:使用位点特异性突变蛋白的体外 AMPK激酶测定。 在野外存在下进行体外激酶测定ype GST-Nrf2或其S558A突变体。

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。

寡肽竞争测定磷酸化位点测定

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

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.4K 次观看.  Seoul National University. 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。 

视频: 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.

用放射性标记的ATP测定蛋白激酶活性

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 ...

科研

生物化学

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.

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

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.

利用多功能生化方法鉴定新型 ck2 激酶基材

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.

使用新激酶蛋白的强生化方法在分子水平上进行表征

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.

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

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.

用于蛋白质磷酸化定量的优化单分子下拉测定

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.

免疫沉淀法研究受体与酶的功能相互作用

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

免疫与感染

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.

植物蛋白复合体的翻译后修饰的鉴定

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

生物学

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.

激酶底物对鉴定使用高通量筛选


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.

比较GTP酶结合蛋白的使用竞争性试验的亲和

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