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?...

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 &amp;ndash; 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>&lt;em&gt;E. coli&lt;/em&gt; 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: &amp;Auml;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 &amp;beta;-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 &amp;alpha; 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 CEN...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

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

18.5K 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

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

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 ...

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 ...

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 &#176;C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 &#177; 0.10 mol/min/mol kinase and the average Km was around 0.5 USA &#181;M at 25 &#176;C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.

This study describes protocols for nonradiometric methods, a bioluminescent ADP detection assay and a phosphate-affinity SDS-PAGE, to determine the kinase activity of cardiac myosin light chain kinase (cMLCK) and the phosphorylation level of its substrate, myosin regulatory light chain (MLC2v).

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

生物学

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

含量测定的LRRK2的激酶活性在体外</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

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 ...

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 ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

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

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