利用多功能生化方法鉴定新型 ck2 激酶基材
Summary
该协议的目的是标记、丰富和识别复杂生物样品 (如细胞裂解物或组织均质) 中的蛋白激酶 ck2 底物。此方法利用 ck2 生物学的独特方面来实现这一目的。
Abstract
对激酶-底物关系的研究对于全面了解这些酶及其下游目标在生理和病理状态下的功能至关重要。ck2 是一种进化保守的血清/苏氨酸激酶, 在多个细胞过程中含有越来越多的数百种底物。由于其各向异性的特性, 识别和表征一套完整的 ck2 基板特别具有挑战性, 仍然是研究这一重要酶的一个障碍。为了应对这一挑战, 我们设计了一种通用的实验策略, 能够有针对性地丰富和识别假定的 ck2 基板。该协议利用了 ck2 独特的双基底物特异性, 允许其底物在细胞或组织裂解液中进行特定的硫代磷酸化。这些底物蛋白随后被烷基化, 免疫沉淀, 并通过液相色谱-串联质谱 (lc-msn) 进行鉴定。我们以前使用这种方法成功地识别 ck2 底物从嗜酸性粒细胞卵巢, 在这里我们扩大了该协议的应用, 人类胶质母细胞瘤细胞, 说明了这种方法的适应性, 以研究这种激酶在各种模型生物和实验系统中的生物学作用。
Introduction
蛋白激酶是信号转导级联的关键成分。这些酶对底物蛋白的磷酸化产生生物反应, 调节控制细胞分裂、代谢和分化等关键事件的关键事件。ck2 是一种普遍表达的嗜酸性血清-苏氨酸激酶, 从酵母保存到人类, 在许多细胞过程中发挥着重要作用, 从转录调节到细胞周期进展到细胞凋亡1 ,2,3。酶是由两个催化α (或α ‘) 亚基和两个调节β亚基4组成的异体体.ck2 除了具有高度的增压外, 还表现出另外两个使其分析复杂化的不寻常特征, 即本构活性5和双共底物特异性 6。后一种特性赋予 ck2 使用 gtp 以及 atp 对底物蛋白进行磷酸化的能力。
小鼠 ck2 催化或调控亚基的遗传缺失导致胚胎死亡, 表明其在发育和器官发生过程中起着至关重要的作用7,8。ck2 在几种癌症中也有过度表达, 因此是一个很有希望的治疗目标9、10、11.事实上, 目前正在为此目的研究针对 ck2 激酶活性的特定抑制剂12、13、14.虽然抑制 ck2 是一个可行的选择, 但鉴于其多向异性, 另一种也许更合理的办法是针对某些癌症进展的关键 ck2 基板。因此, ck2 底物蛋白的综合鉴定和鉴定对于阐明这种激酶在特定组织或肿瘤类型中的特异性功能将具有重要意义。
在这里, 我们描述了一种通用的生化方法, 用于识别 ck2 底物从复杂的生物样本, 如细胞或组织裂解液。该协议利用 ck2 的双共底物特性, 利用 gtp 模拟 gtpγs (鸟苷 5 ‘-[γ-thio] 三磷酸), 而其他内源激酶不能使用。这有效地允许激酶在此样本中 “标记” 其底物, 以便随后进行隔离和识别。
Protocol
注: 确保所需材料可用并做好适当准备 (参见材料表)。 1. 准备工作 机械裂解组织样本 (1-2mg 的组织在100μl 的裂解缓冲液,表 1) 或培养的细胞 (10 厘米板, 在350μl 的裂解缓冲液中融合), 目标是为实验收集总共900μl 的样本。请注意, 此卷稍有超出下面描述的实验所需的内容。 在4°c 条件下, 以 17, 500 x克的离心速度将样品向下旋转3分钟…
Representative Results
图 1提供了实验过程的示意图。该技术的基础是 ck2 在磷基转移中使用 gtp 的异常能力。在外源 ck2 全酶与 gtp 类似物 gtpγs 一起添加到细胞裂解液中, 可导致内源性 ck2 底物的硫代磷酸化。随后用烷基化试剂对苯甲磺酸对硝基苯 (pnbm) 处理裂解液, 在这些特定底物蛋白上生成硫代磷酸酯, 然后使用抗硫代磷酸酯抗体进行免疫沉淀并最终通过质谱技术…
Discussion
在这里, 我们描述了一种相对简单的生化方法来识别复杂生物样本中的蛋白激酶 ck2 底物。该协议的关键步骤是基于 ck2 的不寻常酶特性, 包括 ck2 依赖硫代化特定底物蛋白使用 gtp γ-s 及其随后的免疫沉淀和鉴定。通过这些结果, 我们已经证明了这种方法的效用和多功能性, 因为我们现在已经将这一策略应用于人类胶质母细胞瘤细胞和食子细胞瘤卵巢18。
<p class="jove_…Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了宾夕法尼亚州卫生部向 t. i. s. 提供的英联邦普遍研究增强赠款的部分支持。
Materials
| 12 mg/mL PNBM | Abcam | ab138910 | 40.5 µL |
| 2.5 mM GTPγS | Sigma-Aldrich | G8634-1MG | 5.4 µL |
| Anti-CK2α (E-7) mouse monoclonal antibody | Santa Cruz Biotechnology | sc-373894 | 1:1000 for Western blotting |
| Anti-GAPDH (6C5) mouse monoclonal antibody | Santa Cruz Biotechnology | sc-32233 | 1:1000 for Western blotting |
| Anti-nucleolin rabbit polyclonal antibody | Abcam | ab22758 | 1:1000 for Western blotting |
| Anti-thiophosphate ester [51-8] rabbit monoclonal antibody | Abcam | ab92570 | Varies (final concentration 2.8 µg for each sample) |
| Centrifuge pre-set to 4ºC | ThermoScientific | Sorvall Legend Micro 21R Cat# 75-772-436 | |
| cOmplete Mini EDTA-Free Protease Inhibitor | Roche | 11836170001 | |
| Lysis Buffer | See recipe below | See recipe below | 30 mL |
| Normal rabbit IgG antibody (isotype control) | Cell Signaling Technology | 2729S | Varies (final concentration 2.8 µg for each sample) |
| PD MiniTrap Column | GE Healthcare | 28-9180-10 | 3 columns |
| Protein A/G Plus Agarose Beads | Santa Cruz Biotechnology | sc-2003 | 600 µL |
| Recombinant human CK2 holoenzyme | New England Biolabs | P6010S | 2.7 µL |
| Rotator | Labnet: Mini Labroller | Mini Labroller SKU# H5500 | |
| T98G human glioblastoma cells | ATCC | CRL-1690 | |
| Water bath pre-set to 30ºC | Shel Lab | H20 Bath Series Model# SWB15 |
Riferimenti
- Litchfield, D. W. Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochemical Journal. 369 (Pt 1), 1-15 (2003).
- Ahmed, K., Gerber, D. A., Cochet, C. Joining the cell survival squad: an emerging role for protein kinase CK2). Trends in Cell Biology. 12 (5), 226-230 (2002).
- Meggio, F., Pinna, L. A. One-thousand-and-one substrates of protein kinase CK2. The FASEB Journal. 17 (3), 349-368 (2003).
- Niefind, K., Guerra, B., Ermakowa, I., Issinger, O. G. Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme. The EMBO Journal. 20 (19), 5320-5331 (2001).
- Sarno, S., Ghisellini, P., Pinna, L. A. Unique activation mechanism of protein kinase CK2. The N-terminal segment is essential for constitutive activity of the catalytic subunit but not of the holoenzyme. Journal of Biological Chemistry. 277 (25), 22509-22514 (2002).
- Niefind, K., Putter, M., Guerra, B., Issinger, O. G., Schomburg, D. GTP plus water mimic ATP in the active site of protein kinase CK2. Nature Structural & Molecular Biology. 6 (12), 1100-1103 (1999).
- Lou, D. Y., et al. The alpha catalytic subunit of protein kinase CK2 is required for mouse embryonic development. Molecular and Cellular Biology. 28 (1), 131-139 (2008).
- Buchou, T., et al. Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality. Molecular and Cellular Biology. 23 (3), 908-915 (2003).
- Chua, M. M., et al. CK2 in Cancer: Cellular and Biochemical Mechanisms and Potential Therapeutic Target. Pharmaceuticals (Basel). 10 (1), (2017).
- Tawfic, S., et al. Protein kinase CK2 signal in neoplasia. Histology and Histopathology. 16 (2), 573-582 (2001).
- Hanif, I. M., Hanif, I. M., Shazib, M. A., Ahmad, K. A., Pervaiz, S. Casein Kinase II: an attractive target for anti-cancer drug design. The International Journal of Biochemistry & Cell Biology. 42 (10), 1602-1605 (2010).
- Siddiqui-Jain, A., et al. CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy. Ricerca sul cancro. 70 (24), 10288-10298 (2010).
- Chon, H. J., Bae, K. J., Lee, Y., Kim, J. The casein kinase 2 inhibitor, CX-4945, as an anti-cancer drug in treatment of human hematological malignancies. Frontiers in Pharmacology. 6, 70 (2015).
- Perea, S. E., et al. CIGB-300, a novel proapoptotic peptide that impairs the CK2 phosphorylation and exhibits anticancer properties both in vitro and in vivo. Molecular and Cellular Biochemistry. (1-2), 163-167 (2008).
- Xiao, S., et al. Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation. PLoS One. 9 (10), e109858 (2014).
- Shi, Y., Brown, E. D., Walsh, C. T. Expression of recombinant human casein kinase II and recombinant heat shock protein 90 in Escherichia coli and characterization of their interactions. Proceedings of the National Academy of Sciences of the United States of America. 91 (7), 2767-2771 (1994).
- Mollapour, M., Tsutsumi, S., Kim, Y. S., Trepel, J., Neckers, L. Casein kinase 2 phosphorylation of Hsp90 threonine 22 modulates chaperone function and drug sensitivity. Oncotarget. 2 (5), 407-417 (2011).
- McMillan, E. A., et al. The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis. Journal of Biological Chemistry. 293 (8), 2990-3002 (2018).
- Turowec, J. P., et al. Protein kinase CK2 is a constitutively active enzyme that promotes cell survival: strategies to identify CK2 substrates and manipulate its activity in mammalian cells. Methods in Enzymology. , 471-493 (2010).
- Rusin, S. F., Adamo, M. E., Kettenbach, A. N. Identification of Candidate Casein Kinase 2 Substrates in Mitosis by Quantitative Phosphoproteomics. Frontiers in Cell and Developmental Biologyl. 5, 97 (2017).
- Bian, Y., et al. Global screening of CK2 kinase substrates by an integrated phosphoproteomics workflow. Scientific Reports. 3, 3460 (2013).
- Franchin, C., et al. Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2 inhibitor quinalizarin in HEK-293T cells. Biochimica et Biophysica Acta. 1854 (6), 609-623 (2015).
- Franchin, C., et al. Re-evaluation of protein kinase CK2 pleiotropy: new insights provided by a phosphoproteomics analysis of CK2 knockout cells. Cellular and Molecular Life Sciences. 75 (11), 2011-2026 (2018).
Citazione di questo articolo
Chojnowski, J. E., McMillan, E. A., Strochlic, T. I. Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach. J. Vis. Exp. (144), e59037, doi:10.3791/59037 (2019).