我们感谢加州大学伯克利分校霍华德休斯医学院的大卫. 金博士进行质谱分析和有益的评论。我们感谢雪莲博士, 上海, 中国科学院生物科学研究所, 上海, 中国提供了一个全长的 CENP 建设。最后, 我们感谢苏珊. 贝恩博士和 Christof Grewer 博士在宾汉大学获得设备。这项研究由纽约州立大学和纽约州立大学化学系研究基金会资助。

作者没有什么可透露的。

我们的体外激酶检测是一种非常有效的方法来识别激酶 Cdk1 的分子靶点, 它是细胞周期的主控制器, 调节许多重要的细胞过程。该方法确定纯化蛋白是否为 Cdk1 的基质, 并允许识别特定的磷酸化部位。这促进机械研究通过磷酸化通过 Cdk1 调控细胞过程。
质谱法成功识别磷酸化部位的最关键因素是激酶测定的磷酸化效率。个别场址的磷酸化效率应尽可能高, 最好是100%。这可以通过增加 Cdk1/cyclin B 的数量和增加孵化时间到16小时来实现。在计算所需的 Cdk1/cyclin B 量时, 应考虑蛋白质的摩尔浓度和磷酸化部位的数量。如果目标蛋白中存在多个磷酸化部位, 这一点尤为重要。还应注意的是, 商业 Cdk1/cyclin B 的活动可能会因批次而异, 并且激酶可以迅速地失去活性。如果体外磷酸化不成功, 建议测试激酶的活动。作为对故障诊断的积极控制, 可以使用体外磷酸化 CENP F (残滓 2,987-3, 065)。作为一种负控制, 应执行和分析无 Cdk1/cyclin B 加法的反应。另一个有用的负控制是使用 Cdk1 的激酶死突变体, 这是催化非活动的, 由于一个简单的点突变 (D146N)47,48,49。此外, 为了验证已确定的磷酸化地点, 可以创建一个 phosphonegative 版本的目标蛋白, 在那里磷酸化地点被 alanines 取代。如果确定的磷酸化站点是正确的, 则相应的 phosphonegative 突变体不能被磷酸化。
一个简单的工具, 以检测成功的磷酸化的目标蛋白是简单的 SDS 页凝胶移位化验这里描述。由于增加的大小和负电荷, 磷酸化蛋白在 SDS 页面上的迁移速度通常比 unphosphorylated 的相对较低。请注意, 对于大型蛋白质, 需要优化 SDS 页分析, 以充分提高分辨率以可视化凝胶移位。磷酸-结合标记 (磷标记) 的一个有用的工具为磷酸化蛋白的具体分离。与相应的去磷酸化蛋白50、51相比, 用磷标记丙烯酰胺制备的凝胶中的磷酸化蛋白可视为较慢的迁移带.
为获得高质量的质谱,体外激酶测定中的蛋白质纯度和均匀性是非常重要的。我们的纯化协议通过结合多种高选择性色谱步骤和通过添加蛋白酶抑制剂和还原剂防止蛋白质降解和氧化来实现这一效果。该纯化协议可通过谷胱甘肽 (即) 对蛋白质进行改性, 并洗脱;然而, 应该考虑的是, GST 标签增加了另外 25 kDa, 大蛋白质是挑战质谱的目标。另外, 我们还可以使用其6标记融合蛋白的纯化协议。
还可以修改此协议, 以确定特定于其他激酶的磷酸化站点。唯一的要求是, 纯化激酶是可用的, 活跃的, 并足够稳定。每个激酶的检测条件需要调整, 以达到最大的活性。参数的优化包括激酶的数量, 反应缓冲 (pH 值, 盐浓度), 孵化时间, 反应温度, ATP 浓度。体外激酶检测成功地用于识别许多激酶的磷酸化部位, 包括 Plks (马球样激酶)52,53,54, 和 MAPK/ERK 激酶 (丝裂原活化蛋白激酶/细胞外信号调节激酶)55,56。
虽然这种方法的强度是确定纯化蛋白中的特定磷酸化部位, 但应该指出的是, 作为 Cdk1体外目标, 磷酸化站点不一定是磷酸化在体内。在薇o 中, 还有其他的管理机制, 这可能导致 Cdk1 无法识别的磷酸化站点。例如, 其他交互伙伴可能存在 "体内", 它可以隐藏磷酸化站点或通过结构更改来访问它。此外, 基质需要与 Cdk1/cyclin B 在 G2 阶段的细胞核 colocalize, 以磷酸化。此外, 翻译后的修改或其他管理过程可能会影响目标蛋白体内的构象, 从而使磷酸化站点无法进入。这些限制可以通过在细胞或动物模型中设计适当的实验来克服, 以证实所鉴定的磷酸化部位具有生理功能。在这里, 我们使用一个简单的结合检测功能验证, 因为磷酸化减少了 CENP 和 karyopherin α的相互作用. 还应在细胞或动物模型的上下文中进行功能验证。因此, 我们还转染了 CENP-F 片段的荧光融合蛋白, 其中含有 cNLS 进入 HeLa 细胞10。Phosphomimetic 突变在 CENP 的 cNLS 内减少了核定位, 证实了这些磷酸化点的生理功能10。
有几种工具可用于细胞或动物模型中磷酸化部位的功能验证。Phosphomimetic 突变, 它模仿的影响, 磷酸化的负电荷, 广泛用于这一目的。对于这些, 磷酸化部位 (丝氨酸或苏氨酰) 由天门冬氨酸或谷氨酸取代。其优点是需要对目标蛋白进行点突变。应该指出的是, 虽然在许多情况下成功地使用了 phosphomimetic 突变进行功能验证, 但它们尤其适用于电荷变化是调节的主要因素, 而不是结构变化。在许多其他情况下, Phosphomimetic 突变无法模拟磷酸化的影响 (例如, 11)。然而, 其他方法可用于磷酸化部位的功能验证, 包括使用 phosphonegative 突变: 这些突变通过取代磷酸化部位与丙氨酸来防止磷酸化。在细胞环境中进行功能验证的另一种有用的方法是 Cdk1 的激酶死突变体的表达, 它通过易于引入的点突变 (D146N)47,48,49来灭活。值得注意的是, 一些小分子 Cdk1 抑制剂是商用的, 如 Flavopiridol 和 RO-3306, 可用于细胞或动物模型的功能研究7,8,11。这些抑制剂的特点是良好的, 并有几个在临床试验的癌症治疗 (审查见9)。
虽然在蛋白质组学研究中发现了大量的蛋白磷酸化部位, 但在大多数情况下, 激酶特异性是未知的, 而体外激酶检测是可用于鉴别 Cdk1 基底的几种方法之一。还使用了其他方法。用工程 Cdk1 酶鉴定 Cdk1 靶点。它有一个更大的基板装订袋, 并接受更笨重的版本的 ATP 作为基板6。这种笨重的基质不能与野生型激酶结合。增加核素庞大的 ATP 的细胞提取物含有改良的 Cdk1 酶因此导致特定的标签的直接基质的 Cdk1. 用此方法在芽胞菌中发现 200 Cdk1 靶;然而, 它不能应用于哺乳动物细胞, 这是一个关键的限制。在另一项研究中, 通过使用 Cdk1、Flavopiridol 和 RO-3306 两个小分子抑制剂进行定量 phosphoproteomics 分析, 在人类组织培养细胞中发现 Cdk1 基底。1215磷酸肽持551蛋白在 Cdk1 抑制剂添加7 时显著减少。此方法可以为潜在的 Cdk1 目标筛选整个蛋白质组, 但需要通过体外激酶检测来验证所产生的目标,例如。
将来,体外激酶检测很可能与目前正在开发的 Cdk1 基板的高通量屏幕结合在一起。由于蛋白质组中大量的 Cdk1 基质, 许多人类 Cdk1 基质仍有待识别, 而体外激酶检测将是识别、映射和验证这些站点的重要工具。
体外激酶检测是确定激酶 Cdk1 的靶点和确定特定磷酸化部位的有力工具。这些磷酸化部位的鉴定, 使机械学研究, 以调节细胞过程的 Cdk1。

激酶是将磷酸酯从 ATP 转移到基质并调节许多细胞过程的酶。这种磷酸化是可逆的, 快速的, 增加两个负电荷, 并储存自由的能量, 是最常见的翻译后修改之一, 细胞使用。Cdk1, 也称为细胞分裂周期蛋白2同源 (cdc2) 是所有真核生物的细胞周期的主控制器1,2,3,4,5, 和 phosphorylates 一个估计8-13% 的蛋白质组6,7。
虽然最近的蛋白质组研究已经确定了许多蛋白质磷酸化部位, 在大多数情况下, 负责这些修改的激酶是未知的。已知的 Cdk1 目标 (特别是在人类细胞中) 的数量低7。Cdk1-specific 磷酸化部位的鉴定是很重要的, 因为它能使机械学研究确定 Cdk1 如何控制细胞周期。细胞周期调节对于忠实的染色体分离和细胞分裂是很重要的, 需要大量的细胞过程来支持这个重要的生理功能。这包括在有丝分裂开始前停止转录和翻译, 以及细胞结构和组织的戏剧性重组, 如核包膜的拆卸、染色体凝结和有丝分裂纺锤体的组装。这些过程中的放松管制和错误导致癌症、出生缺陷或有丝分裂细胞死亡。Cdk1 的特定抑制剂如 RO-3306 被开发了8, 为功能性研究提供强有力的工具, 并且其中一些抑制剂当前在临床试验为癌症治疗 (参见9为审查)。
在这里, 我们描述了一个体外激酶检测, 允许鉴定 Cdk1-specific 磷酸化部位。在这项试验中, 商业上可用的人类 Cdk1/cyclin B 用于 phosphorylate 纯化的目标蛋白体外。基板的磷酸化增加了其质量, 增加了两个负电荷;因此, 蛋白凝胶带在 SDS 页上的向上转移证实了成功的磷酸化。Cdk1-specific 磷酸化部位随后通过质谱分析对体外磷酸化蛋白进行鉴定。为了辅助实验设计, 我们还回顾了从蛋白质序列预测 Cdk1-specific 磷酸化部位的计算工具和参考文献。此外, 我们还描述了纯化的协议, 产生高纯度和同类蛋白制剂适合的激酶检测。最后, 确定的磷酸化部位必须经功能研究证实, 并在此描述一个简单的结合试验。结合, 这是一个非常强大的方法, 产生 Cdk1-specific 磷酸化网站, 并使机械研究如何 Cdk1 控制细胞周期7,10,11。由于这种方法依赖于纯化的蛋白质, 它可以应用于任何模型有机体, 并产生可靠的结果。但是, 建议对获得的磷酸化站点体外进行功能验证, 因为细胞有额外的调控机制, 如翻译后修改、交互伙伴或细胞定位,可能使磷酸化的网站容易被 Cdk1 承认或无法获得。
Cdk1 认识到一个协商一致的磷酸化站点, 它由 (x-赖氨酸/精氨酸) 组成, 其中 x-是任何残留物, 而丝氨酸或苏化是磷酸化的场所。特别重要的是要识别的是存在的脯氨酸在 +1 的位置。此外, 在 +2 或 +3 位中, 基本残留物是首选的, 大多数 Cdk1-specific 磷酸化站点含有赖氨酸或 Arg, 在 +3 位置6,12。
激活 Cdk1 被严密地调控并且导致有丝分裂的开始1,2,3,4,5。细胞周期蛋白依赖性激酶的活动一般取决于它们与不同的细胞周期蛋白 (周期蛋白 A、B、C、D 和 E 在人类中) 的关联, 这些都是以振荡的水平表达的, 在整个单元循环中13。Cdk1 的表达是恒定的在细胞周期和它的活动的章程依靠它与调控亚基周期蛋白 A 和细胞周期蛋白 B5,13,14,15的关联, 作为以及翻译后的修改。Cdk1/cyclin B 复合体的形成需要为激酶激活5,14,15,16,17,18。在 G2 阶段, 细胞周期蛋白 B 在细胞质中被翻译并导入到细胞核中, 它绑定到 Cdk15,14,15,16,17,18;然而, Cdk1/cyclin B 是由人类 Cdk1-inhibitory 激酶 Myt1 (与膜相关的酪氨酸-和苏氨酸特定 cdc2-inhibitory 激酶) 和 Wee1 分别在残留 Thr14 和 Tyr15 上的磷酸化而被灭活而成的19, 20,21。在晚期 G2 阶段, 细胞分裂周期25磷酸酶 (cdc25) 除磷 Thr14 和 Tyr15 激活 Cdk1/cyclin B 复合体的激酶活性并触发有丝分裂 12, 14,18,20,22,23. Thr161 的磷酸化也需要 Cdk1/cyclin B 活化, 并由 Cdk7、Cdk 活化激酶 (CAK)18介导。细胞周期蛋白 B 在后期钝化 Cdk1 中的降解, 允许从有丝分裂中退出24,25。因此, 活化 Cdk1/cyclin B 是一个复杂的过程。此处介绍的协议是用商用 Cdk1/cyclin B 进行的。在这种复杂的昆虫细胞的重组表达, 它是激活在体内由内源性激酶14,20 , 并保持活跃的纯化状态。所产生的活性, 重组人 Cdk1/cyclin B 适用于体外激酶测定。
在这里, 我们描述了在人类着丝粒蛋白 F (CENP)10 中鉴定 Cdk1-specific 磷酸化点的协议。CENP 是一种动粒蛋白, 驻留在细胞核中的大部分界面 (G1 和 s-阶段), 并导出到细胞质 G2 阶段 26, 27, 28 在 Cdk1-dependent 方式 10, 11. 核本地化由二分 cNLS26授予。cNLSs 由核运输因素 karyopherin α认可, 促进, 与 karyopherin β和 RanGDP 一起, 进口 cNLS-货物入核29。G2 阶段的核出口是通过未知的出口途径10来促进的。一旦 CENP 驻留在细胞质, 它被招募到核信封, 并反过来招募马达蛋白复合蛋白30,31。这一途径是重要的, 以蛋白的方式在有丝分裂纺锤体的初始阶段定位细胞核各自的中心体, 这对于正确的有丝分裂时机和大脑的基本过程是重要的。开发30,31,32。从 G2 阶段开始, CENP 也被组装到动粒中, 它对忠实的染色体隔离有重要的作用 27, 28, 33, 34, 35.这些路径的一个关键管理步骤是在 G2 阶段 CENP 的核输出, 它依赖于 Cdk110、11。这里我们描述了一个在 CENP cNLS 的 Cdk1-specific 磷酸化部位鉴定的协议。这些站点的 Phosphomimetic 突变减慢了 CENP 的核进口, 这表明 Cdk1/cyclin B 通过其 cNLS10的磷酸化直接调节 CENP F 的细胞定位。
总体而言, 此体外激酶检测允许识别激酶 Cdk1 的特定基底. 纯化靶蛋白是磷酸化体外由商业可利用的 Cdk1/cyclin B 复合体和磷酸化点随后用质谱法鉴定。Cdk1-specific 磷酸化部位的鉴定支持机械研究, 揭示了 Cdk1 如何控制细胞周期。

<table>
	<tbody>
		<tr>
			<td>2800 ml baffled Fernbach flask</td>
			<td>Corning</td>
			<td>44232XL</td>
			<td></td>
		</tr>
		<tr>
			<td>ampicillin</td>
			<td>Gold Biotechnology</td>
			<td>A-301-25</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Fisher Scientfiic</td>
			<td>BP413-25</td>
			<td></td>
		</tr>
		<tr>
			<td>benzamidine hydrochloride</td>
			<td>Millipore Sigma</td>
			<td>B6506-25</td>
			<td></td>
		</tr>
		<tr>
			<td>bottletop filter</td>
			<td>Corning</td>
			<td>431161</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B recombinant, human 20,000 U/mL</td>
			<td>New England Biolabs</td>
			<td>P6020</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B (alternate source)</td>
			<td>EMD Millipore</td>
			<td>14-450</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B (alternate source)</td>
			<td>Invitrogen</td>
			<td>PV3292</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/cyclin B + 10x PK buffer</td>
			<td>New England Biolabs</td>
			<td>P6020</td>
			<td></td>
		</tr>
		<tr>
			<td>CENP-F (residues 2987 &#8211; 3065) pGEX6P1 plasmid</td>
			<td>Available upon request.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge: Heraeus Multifuge X3R, cooled, with TX-1000 swing-out rotor</td>
			<td>Thermo Scientific</td>
			<td>10033-778</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifugal filter units: Amicon Ultra-15 centrifugal filter units, 3 kDa cutoff, Ultracel-PL membranes</td>
			<td>EMD Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>chlorampenicol</td>
			<td>Gold Biotechnology</td>
			<td>C-105-100</td>
			<td></td>
		</tr>
		<tr>
			<td>D/L methionine</td>
			<td>Agros Organics / Fisher</td>
			<td>125650010</td>
			<td></td>
		</tr>
		<tr>
			<td>desalting pipet tips: Zip tips</td>
			<td>Millipore Sigma</td>
			<td>ZTC18S008</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable chromatography columns, Econo-Pac 1.5 x 12 cm</td>
			<td>Biorad</td>
			<td>7321010</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Gold Biotechnology</td>
			<td>DTT50</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli Rosetta 2(DE3)pLysS strain</td>
			<td>EMD Millipore</td>
			<td>71403</td>
			<td></td>
		</tr>
		<tr>
			<td>electrospray ionization Fourier transform ion</td>
			<td>Bruker Amazon</td>
			<td>Apex III</td>
			<td></td>
		</tr>
		<tr>
			<td>cyclotron resonance mass spectrometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>electrospray ionization ion trap mass spectrometer</td>
			<td>Bruker Amazon</td>
			<td>custom</td>
			<td></td>
		</tr>
		<tr>
			<td>fixed angle rotor: Fiberlite F15-8x-50cy</td>
			<td>Thermo Scientific</td>
			<td>97040-276</td>
			<td></td>
		</tr>
		<tr>
			<td>FPLC system: &#196;kta Pure FPLC</td>
			<td>GE Healthcare</td>
			<td>29032697</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel filtration column: Superdex 200 Increase 10/300 GL</td>
			<td>GE Healthcare</td>
			<td>28990944</td>
			<td></td>
		</tr>
		<tr>
			<td>glutathione agarose</td>
			<td>Pierce</td>
			<td>16101</td>
			<td></td>
		</tr>
		<tr>
			<td>glutathione, reduced</td>
			<td>Millipore Sigma</td>
			<td>G4251-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>incubation shaker: multitron shaker</td>
			<td>Infors</td>
			<td>I10102</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Gold Biotechnology</td>
			<td>I2481C50</td>
			<td></td>
		</tr>
		<tr>
			<td>kanamycin</td>
			<td>Gold Biotechnology</td>
			<td>K-120-25</td>
			<td></td>
		</tr>
		<tr>
			<td>karyopherin &#945; pet-28a pres plasmid</td>
			<td>Available upon request.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani medium</td>
			<td>Fisher Scientfiic</td>
			<td>BP1426-2</td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge 5418R, refrigerated</td>
			<td>Eppendorf</td>
			<td>5401000013</td>
			<td></td>
		</tr>
		<tr>
			<td>microtubes (0.5 ml)</td>
			<td>Eppendorf</td>
			<td>30121023</td>
			<td></td>
		</tr>
		<tr>
			<td>microtubes (1.5 ml)</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel affinity gel: His-Select Nickel affinity gel</td>
			<td>Millipore Sigma</td>
			<td>P6611-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEX-6P-1 plasmid</td>
			<td>Millipore Sigma</td>
			<td>GE28-9546-48</td>
			<td></td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride</td>
			<td>Gold Biotechnology</td>
			<td>P470-10</td>
			<td></td>
		</tr>
		<tr>
			<td>PS protease: PreScission protease</td>
			<td>GE Healthcare</td>
			<td>27084301</td>
			<td></td>
		</tr>
		<tr>
			<td>Phos-tag acrylamide</td>
			<td>Wako Pure Chem. Ind.</td>
			<td>304-93521</td>
			<td></td>
		</tr>
		<tr>
			<td>reduced gluthathione</td>
			<td>Millipore Sigma</td>
			<td>G4251-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>roundbottom centrifuge tubes (Oakridge tubes)</td>
			<td>Fisher Scientfiic</td>
			<td>055291D</td>
			<td></td>
		</tr>
		<tr>
			<td>site-directed mutagenesis kit: QuikChange Lightning</td>
			<td>Agilent</td>
			<td>210518</td>
			<td></td>
		</tr>
		<tr>
			<td>Site-Directed Mutagenesis Kit</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sonifier: Branson S-250D sonifier</td>
			<td>Branson</td>
			<td>15 338 125</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectra/Por 1RC dialysis membrane (6-8 kDa cutoff)</td>
			<td>Spectrum Labs</td>
			<td>08 670B</td>
			<td></td>
		</tr>
		<tr>
			<td>swing out rotor TX-1000</td>
			<td>Thermo Scientific</td>
			<td>10033-778</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of cyclin-dependent kinase 1 specific phosphorylation sites by an in vitro kinase assay

周期蛋白依赖性激酶 1 (Cdk1) 是一个主控制器为细胞周期在所有真核生物和 phosphorylates 估计 8-13% 的蛋白质组;然而, 已确定的 Cdk1 目标的数目, 特别是在人类细胞中的数量仍然很低。Cdk1-specific 磷酸化部位的鉴定是重要的, 因为它们提供了机械的洞察力如何 Cdk1 控制细胞周期。细胞周期调节对忠实的染色体分离至关重要, 而在这个复杂过程中的缺陷导致染色体畸变和癌症。
在这里, 我们描述了一个体外激酶检测, 用于识别 Cdk1-specific 磷酸化部位。在本试验中, 纯化蛋白是通过商业上可获得的人类 Cdk1/cyclin 磷酸化而成的体外b. 成功的磷酸酶是由 SDS 页确认的, 并且随后用质谱法鉴定磷酸的部位。我们还描述了纯化的协议, 产生高纯度和均匀的蛋白质制剂, 适合于激酶的检测, 和结合试验的功能验证鉴定的磷酸化部位, 探讨之间的相互作用经典核定位信号 (cNLS) 及其核传输受体 karyopherin α。为了辅助实验设计, 我们回顾了从蛋白质序列预测 Cdk1-specific 磷酸化部位的方法。这些协议一起提供了一个非常强大的方法, 产生 Cdk1-specific 磷酸化网站, 并使机械研究如何 Cdk1 控制细胞周期。由于这种方法依赖于纯化的蛋白质, 它可以应用于任何模型有机体和产生可靠的结果, 特别是当结合细胞功能研究。

我们最近使用了体外激酶检测 (图 1) 来确定包含 cNLS10的 CENP 片段中的 Cdk1-specific 磷酸化站点。这一信号赋予 CENP 在界面的核定位。在 G2 阶段, CENP 以 Cdk1-dependent 的方式从原子核向细胞质出口。为了获得关于 Cdk1 如何调节 CENP 细胞定位的机械见解, 我们通过 iGPS 服务器36,37, 分析了 CENP 的序列, 以预测 Cdk1-specific 磷酸化站点。在 CENP 的 cNLS 中, 三个 Cdk1-specific 磷酸化部位在残滓3042、3045和 3048 (表 1) 中被预测。另一个 Cdk1-specific 磷酸化站点也被预测为残滓 3007 (表 1)10。因此, 我们假设 Cdk1 直接通过其 cNLS 的磷酸化来调节 CENP 的定位。
为了测试 CENP 的 cNLS 是否为 Cdk1 的基板, 我们用纯化的人体 CENP F 片段 (残滓2,987-3、065) 和人类 Cdk1/cyclin B 进行了体外激酶检测.体外磷酸化 CENP-f 在 16% SDS 页上进行了分析,与缺少激酶 Cdk1 (图 2A)10的负控制一起使用。对于体外磷酸化 CENP, 与负控制相比, 在凝胶上观察到带的明显向上移位。这是典型的磷酸化蛋白, 因为每个磷酸盐组增加两个负电荷和额外的质量。这些结果表明, CENP 是磷酸化的 Cdk1/cyclin B10。
通过质谱 (ESI-ITMS)10分析, 确定 CENP 磷酸化部位的数量, 并定量测定这些部位的增殖效率, 并对完整的体外磷酸化 CENP 片段 (84) 进行了研究。观测到的 ESI-离子阱质谱 (图 2B) 表明, 完整的 CENP 片段是磷酸化在四个站点 (表 1)10。
通过对胰蛋白酶和 arginines 后以及蛋白链的研究, 对 CENP 序列中磷酸化氨基酸部位的定位进行了评估, 并对 CENP F 片段的胰蛋白酶文摘进行了若干多肽, 包括含有残留物 3,031-3, 052 的一种。此 CENP 肽的质谱与在 cNLS (表 1)10中的残留 T3042、T3045 和 S3048 的磷酸化相一致。另一胰蛋白酶肽 (残滓2,995-3、016) 的质谱与 S3007 (表 1)10中的磷酸化一致。所有四 Cdk1-specific 磷酸化部位也被预测从序列。Cdk1-specific 基板通常含有在磷酸化6的残留物旁边的脯氨酸, 如残留物 T3042、T3045 和 S3007 所观察到的。应该指出的是, S3048 是一个有点不寻常的 Cdk1-specific 磷酸化站点, 因为苯丙氨酸位于丝氨酸旁边。最后, 结果表明, CENP 是在残留物 3007, 3042, 3045, 和3048的 Cdk1 的激酶, 特别是磷酸化。其中三个残留物位于 CENP F (表 1) 的 cNLS 中, 表明 Cdk1 可能通过 cNLS 阶段的 G2 的磷酸化来调节 CENP 细胞的定位, 当 Cdk1 变为活动10时。
为了验证这些 Cdk1-specific 磷酸化站点是否具有生理功能, 我们创建了 phosphomimetic 变体 S3048D10。Phosphomimetic 突变通过负电荷残留物模拟磷酸化的影响。接下来, 我们纯化了包含 cNLS 的 CENP 片段的 phosphomimetic 版本。CENP 的 cNLS 被核转运受体 karyopherin α所识别, 它具有很强的亲和性, 并且是 CENP F 的核进口所必需的。为了测试 phosphomimetic 突变是否削弱了 CENP cNLS 与其核传输受体 karyopherin α之间的相互作用, 我们进行了绑定检测 (图 3)10。在这些实验中, 纯化的 CENP 片段 (野生型或 S3048D 变种) 与纯化的人 karyopherin α混合, 并用解析尺寸排除色谱分离出混合物。此外, 还对蛋白质进行了单独的分析。对洗脱分数的 SDS 页分析发现, 野生型 CENP-F 片段与 cNLS 强烈地相互作用与 karyopherin α, 因为两个蛋白质共同洗脱 (图3) 10.然而, 只有微不足道的 CENP S3048D 片段绑定到 karyopherin α, 这些蛋白质洗脱单独 (图3) 10.这些结果表明, CENP cNLS 3048 的磷酸化对与核转运因子 karyopherin α的相互作用有很强的影响。应当指出, 核进口率高度依赖核定位信号对核运输因子46的亲和力, 因此, 预计这些突变会减慢核进口的10。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57674/57674fig1.jpg"> 图 1: Cdk1 的示意图表示法体外激酶测定.<a href="/files/ftp_upload/57674/57674fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57674/57674fig2.jpg"> 图 2: CENP 是由 Cdk1/cyclin B 磷酸化的.(A) 确定 Cdk1 特定磷酸化部位, 用纯化的 CENP (残留物 2,987-3, 065) 进行了体外激酶测定。对无 Cdk1/cyclin B 的负控制的 SDS 页分析显示在左车道 (-Cdk1), 旁边的体外磷酸化 CENP 在右车道 (+ Cdk1)。指出了分子量标准。注意磷酸化 CENP 在 SDS 页上的向上转移, 这与成功磷酸化是一致的。Cdk1 和细胞周期蛋白 B 在 34 kDa 和 60 kDa 中呈微弱的带状。(B) 用 CENP (A) 中完整磷酸化的离子阱质谱法测定了完整磷酸化 CENP (84) 的磷酸盐负荷。并给出了相应的质量谱。强度的绘制与质量-电荷比 (m/z)。光谱显示了磷酸化后完整的 CENP 片段的 +10 到 +14 电荷状态。它们表明有0、1、2、3和4磷酸酯的物种种群。为了量化每个物种与总蛋白质数量的比值, 确定并比较了峰值高度 (表 1)。此数字已从10中修改, 并在泰勒和弗朗西斯出版商的许可下复制。<a href="/files/ftp_upload/57674/57674fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57674/57674fig3.jpg"> 图 3: 功能验证: CENP F 的 phosphomimetic 突变强烈地减少了与核转运受体 karyopherin α的相互作用.(A-F)纯化的 CENP 片段 (残留物 2,987-3, 065) (0.1 毫克) 和纯化的 karyopherin α (0.7 毫克) 和混合在1:1 摩尔比和分析的大小排除色谱。CENP 片段和 karyopherin α也分别进行了分析。给出了峰值分数的 SDS 页分析。洗脱量在底部标明 (以0.6 毫升递增)。在左侧, kDa 分子量标记带及其质量的位置。星号标记残留 GST (谷胱甘肽 S 转移酶) 的痕迹。(A) Karyopherin α (B) 洗脱型材。在 280 nm 的吸光度与洗脱量绘制。karyopherin α (红色) 的洗脱剖面用 karyopherin α和 CENP 的混合物的洗脱剖面覆盖 (野生型: 绿色; phosphomimetic S3048D 突变体: 蓝色)。注意, 野生型 CENP 片段的加入将 karyopherin α峰的洗脱量转移到更高的质量, 这与 CENP 野生型片段的结合是一致的。当添加 phosphomimetic 突变 S3048D 的 CENP 片段时, 没有观察到这种变化, 这表明 phosphomimetic 突变极大地减少了 CENP f 与 karyopherin α的相互作用. (C) CENP f 野生类型片段 (小波) 和karyopherin α. (D) CENP 片段。(E) CENP S3048D 片段和 karyopherin α. (F) CENP F S3048D 片段。此数字是用10中的数据创建的。<a href="/files/ftp_upload/57674/57674fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="3">表1A。CENP 片段的序列 (残滓 2987-3065) 与预测的 Cdk1-specific 磷酸化站点以更大的字体大小突出显示。胰蛋白酶片段以粗体和带下划线突出显示。</td>
		</tr>
<tr>
<td colspan="3">GPLGSQQSKQDSRGSPLLGPVVPGPSPIPSVTEKRLSSGQNKAS</td>
		</tr>
<tr>
<td colspan="3">GKRQRSSGIWENGGGPTPATPESFSKKSKKAVMSGIHPAE</td>
		</tr>
<tr>
<td colspan="3">表1B。完整 CENP 的质谱分析</td>
		</tr>
<tr>
<td colspan="3">84在体外磷酸化后的 Cdk1/cyclin B</td>
		</tr>
<tr>
<td></td>
			<td>检测到的磷酸化站点 (P)</td>
			<td>磷酸盐负荷以总蛋白% 表示</td>
		</tr>
<tr>
<td rowspan="5">CENP-F 2987-3065</td>
			<td>0P</td>
			<td>6%</td>
		</tr>
<tr>
<td>1P</td>
			<td>37%</td>
		</tr>
<tr>
<td>2P</td>
			<td>40%</td>
		</tr>
<tr>
<td>3P</td>
			<td>15%</td>
		</tr>
<tr>
<td>4P</td>
			<td>3%</td>
		</tr>
<tr>
<td colspan="3">表1C。Cdk1/cyclin B CENP体外磷酸化后胰蛋白酶肽的质谱分析</td>
		</tr>
<tr>
<td></td>
			<td>磷酸化站点的 #</td>
			<td>磷酸盐负荷</td>
		</tr>
<tr>
<td>胰蛋白酶磷酸,</td>
			<td>0P</td>
			<td>57%</td>
		</tr>
<tr>
<td>残渣2995-3016</td>
			<td>1P</td>
			<td>43%</td>
		</tr>
<tr>
<td>胰蛋白酶磷酸,</td>
			<td>0P</td>
			<td>7%</td>
		</tr>
<tr>
<td>残渣3031-3052</td>
			<td>1P</td>
			<td>64%</td>
		</tr>
<tr>
<td></td>
			<td>2P</td>
			<td>29%</td>
		</tr>
<tr>
<td></td>
			<td>3P</td>
			<td>1%</td>
		</tr>
</tbody></table>表 1: Cdk1/cyclin B.在体外磷酸化后 CENP 片段的质谱分析.此表是用10中的数据创建的。

Watch this Scientific Journal Video about Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay at JoVE.com

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

细胞周期蛋白依赖性激酶 1 (Cdk1) 在 G2 阶段被激活并且调控许多细胞通路。在这里, 我们提出了一个协议的体外激酶检测与 Cdk1, 这使得识别 Cdk1-specific 磷酸化的网站, 以建立细胞目标的这个重要的激酶。

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-cyclin-dependent-kinase-1-specific-phosphorylation

Nanyang Technological University

Research

JoVE Journal

Biochemistry

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

18.4K 次观看.  State University of New York at Binghamton. 细胞周期蛋白依赖性激酶 1 (Cdk1) 在 G2 阶段被激活并且调控许多细胞通路。在这里, 我们提出了一个协议的体外激酶检测与 Cdk1, 这使得识别 Cdk1-specific 磷酸化的网站, 以建立细胞目标的这个重要的激酶。

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

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.

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

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

科研

生物化学

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 ...

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.

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.

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 ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

人类外周血神经细胞分离通过评估Rab10磷酸化来询问帕金森氏症相关的LRRK2激酶途径

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

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 ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

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.

通过对Rho激酶活性的暂时抑制,增强人类诱导多能干细胞衍生心肌细胞的诱导

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.

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 ...

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.

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...