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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 2800 ml baffled Fernbach flask | Corning | 44232XL | |
| ampicillin | Gold Biotechnology | A-301-25 | |
| ATP | Fisher Scientfiic | BP413-25 | |
| benzamidine hydrochloride | Millipore Sigma | B6506-25 | |
| bottletop filter | Corning | 431161 | |
| Cdk1/cyclin B recombinant, human 20,000 U/mL | New England Biolabs | P6020 | |
| Cdk1/cyclin B (alternate source) | EMD Millipore | 14-450 | |
| Cdk1/cyclin B (alternate source) | Invitrogen | PV3292 | |
| Cdk1/cyclin B + 10x PK buffer | New England Biolabs | P6020 | |
| CENP-F (residues 2987 – 3065) pGEX6P1 plasmid | Available upon request. | ||
| centrifuge: Heraeus Multifuge X3R, cooled, with TX-1000 swing-out rotor | Thermo Scientific | 10033-778 | |
| centrifugal filter units: Amicon Ultra-15 centrifugal filter units, 3 kDa cutoff, Ultracel-PL membranes | EMD Millipore | UFC900324 | |
| chlorampenicol | Gold Biotechnology | C-105-100 | |
| D/L methionine | Agros Organics / Fisher | 125650010 | |
| desalting pipet tips: Zip tips | Millipore Sigma | ZTC18S008 | |
| disposable chromatography columns, Econo-Pac 1.5 x 12 cm | Biorad | 7321010 | |
| dithiothreitol | Gold Biotechnology | DTT50 | |
| E. coli Rosetta 2(DE3)pLysS strain | EMD Millipore | 71403 | |
| electrospray ionization Fourier transform ion | Bruker Amazon | Apex III | |
| cyclotron resonance mass spectrometer | |||
| electrospray ionization ion trap mass spectrometer | Bruker Amazon | custom | |
| fixed angle rotor: Fiberlite F15-8x-50cy | Thermo Scientific | 97040-276 | |
| FPLC system: Äkta Pure FPLC | GE Healthcare | 29032697 | |
| Gel filtration column: Superdex 200 Increase 10/300 GL | GE Healthcare | 28990944 | |
| glutathione agarose | Pierce | 16101 | |
| glutathione, reduced | Millipore Sigma | G4251-50g | |
| incubation shaker: multitron shaker | Infors | I10102 | |
| isopropyl β-D-1-thiogalactopyranoside | Gold Biotechnology | I2481C50 | |
| kanamycin | Gold Biotechnology | K-120-25 | |
| karyopherin α pet-28a pres plasmid | Available upon request. | ||
| Luria Bertani medium | Fisher Scientfiic | BP1426-2 | |
| microcentrifuge 5418R, refrigerated | Eppendorf | 5401000013 | |
| microtubes (0.5 ml) | Eppendorf | 30121023 | |
| microtubes (1.5 ml) | Eppendorf | 30120086 | |
| Nickel affinity gel: His-Select Nickel affinity gel | Millipore Sigma | P6611-100ml | |
| pGEX-6P-1 plasmid | Millipore Sigma | GE28-9546-48 | |
| phenylmethylsulfonyl fluoride | Gold Biotechnology | P470-10 | |
| PS protease: PreScission protease | GE Healthcare | 27084301 | |
| Phos-tag acrylamide | Wako Pure Chem. Ind. | 304-93521 | |
| reduced gluthathione | Millipore Sigma | G4251-50g | |
| roundbottom centrifuge tubes (Oakridge tubes) | Fisher Scientfiic | 055291D | |
| site-directed mutagenesis kit: QuikChange Lightning | Agilent | 210518 | |
| Site-Directed Mutagenesis Kit | |||
| sonifier: Branson S-250D sonifier | Branson | 15 338 125 | |
| Spectra/Por 1RC dialysis membrane (6-8 kDa cutoff) | Spectrum Labs | 08 670B | |
| swing out rotor TX-1000 | Thermo Scientific | 10033-778 |
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