激酶底物对鉴定使用高通量筛选
Summary
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.
Abstract
我们已经开发出一种筛选平台，以确定专用人蛋白激酶磷酸化的底物，可用于阐明新的信号转导途径。我们的方法的特征是使用纯化的GST标记的人蛋白激酶的文库和感兴趣的重组蛋白衬底。我们已经用这种技术来识别的MAP /微管亲合性 – 调节激酶2（MARK2）作为激酶的葡萄糖调节站点上CREB稳压转录辅激活2（CRTC2），所需的β细胞增殖的蛋白质，以及AXL家族酪氨酸激酶的作为细胞转移的磷酸化的衔接蛋白ELMO的调节剂。我们描述这种技术，并讨论如何帮助建立细胞对环境刺激如何应对一个全面的地图。
Introduction
蛋白质的翻译后修饰（翻译后修饰）是细胞内的通信是必不可少的。也许最好的研究了所有翻译后修饰是磷酸化，由蛋白激酶，从而调节的蛋白质的功能，包括它们的生化活性，亚细胞定位，构象和稳定性无数催化。关于靶蛋白磷酸化位点的鉴定可以通过胰蛋白酶磷酸肽映射或通过使用富含磷酸化肽1,2-样品现在标准蛋白质组技术来完成。而四分之三的表达蛋白质组预计被磷酸和3所识别200000磷酸化位点5，据估计高达1 亿 6，许多这些没有分配的生物学，信号传导途径，或蛋白激酶。
虽然识别磷酸化位点是相对简单的，相对更大的挑战是，鉴定同源激酶（s）表示，针对这些网站，我们称为映射激酶的方法：基片对。几种方法用于鉴定激酶：基板对已经描述，无论是在开始用感兴趣的激酶和寻找其底物或开始与感兴趣的基板，并试图找到改性激酶实验7-11或计算12。为了鉴定激酶为已知的磷酸化底物，生物信息学可以用来识别包含氨基酸侧翼磷酸化残基（共识位点），以及识别形成沉淀络合物与基板激酶短保守序列的蛋白质。然而，这些方法是耗时，经常不与成功。
我们开发了一个系统功能的方法来迅速查明激酶磷酸化可以在给定基片13。屏幕试验产生优良的比性，具有非常明确的选择潜在的同源激酶。鉴于磷酸化生物信号的中心地位，画面中几乎所有的细胞信号通路14-16用于发现有用的。屏幕包括与人蛋白激酶的文库进行大规模激酶测定。所述激酶已被标记为细菌谷胱甘肽-S-转移酶（GST）蛋白和从哺乳动物细胞提取物纯化的，这意味着该重组酶 – 不同于那些来自细菌制备 – 将在上游蛋白激酶经常所需的存在而产生重组酶具有体外活性。实际上，虽然所需的下游激酶活化丝氨酸，苏氨酸，和酪氨酸激酶活性是存在于酵母10中，酵母基因组编码122蛋白激酶，指示所述哺乳动物激酶组，有超过500个基因17，已成为显著更加复杂，以边条忒独特高阶生物体的过程。此外，相关的细胞生物学和人类疾病（如小分子，生长因子，激素， 等等）不同的刺激的效果可以用来14,15调节激酶活性在一个适当的范围内。
Protocol
Representative Results
Discussion
由于原来的出版物描述的方法14,15，180 GST-激酶原库已扩大到420件，或约80％的人类蛋白质激酶组的。与膨胀的文库，如所描述的协议需要4-5天，然后1-4天来开发薄膜（认为必要），这可以通过使用磷光和数字信号增强的缩短。有些情况必须注意几个关键步骤（ 见图 1协议的概述）。第一，是细胞的股票的健康（无支原体，决不允许在膨胀过程中达到汇合，并用胰蛋白酶在每次…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由NSERC经费资助 386634.我们想感谢Screaton实验室成员，有益的讨论。
Materials
| Lysis buffer | Made in house | See Protocol step 1.1 | |
| 10x kinase buffer | Made in house | See Protocol step 1.2 | |
| 10x M-ATP | Made in house | See Protocol step 1.3 | |
| Human kinase plasmids | Orfeome, Invitrogen, Origene | GST-tagged in house | |
| 96 well plates | Fisher Scientific | CS003595 | |
| 293T cells | ATCC | CRL-11268 | |
| DMEM | Fisher Scientific | SH3002201 | supplement with 100U/ml penicillin, 100ug/ml streptomycin, 10% fetal calf serum. |
| CO2 incubator | Sanyo | MCO-17AIC | |
| 15 cm cell culture dishes | Fisher Scientific | 877224 | |
| Reduced serum medium | Invitrogen | 22600-050 | |
| Lipid-based transfection reagent | Invitrogen | 11668-019 | |
| Automated liquid dispenser | Thermo Scientific | 5840300 | |
| Small cassette attachment | Thermo Scientific | 24073295 | |
| Standard cassette attachment | Thermo Scientific | 14072670 | |
| 4mM pervanadate | Made in house | See Protocol step 3.1 | |
| 0.25 M CaCl2 | Made in house | ||
| Multichannel pipette (20-200 uL) | Labnet | p4812-200 | |
| Multichannel pipette (1-10 uL) | Thermo Scientific | 4661040 | |
| V-bottom 6-well plates | Evergreen Scientific | 290-8116-01V | |
| Glutathione coated 96-well plates | Fisher Scientific | PI-15240 | |
| Hybridization oven | Biostad | 350355 | |
| GST tagged substrate | Made in house | ||
| Myelin Basic Protein (MBP) | Sigma | M1891 | |
| Repeater pipette (1 mL) | Eppendorf | 22266209 | |
| 32P gamma-ATP | Perkin Elmer | BLU502Z500UC | |
| 2X SDS lysis buffer (100 mL) | Made in house | See Protocol step 1.4 | |
| 26-well precast TGX gels | BioRad | 567-1045 | gel percentage required is dependent on the molecular weight of the substrate of interest |
| Coomassie stain | Made in house | 0.1% Coomassie R250, 10% acetic acid, 40% methanol | |
| Coomassie destain | Made in house | 10% acetic acid, 20% methanol | |
| Labeled gel containers | Made in house | Used plastic lids from empty tip boxes, just big enough to contain one gel | |
| Whatman filter paper | Fisher Scientific | 57144 | |
| Cellophane sheets (2) | BioRad | 165-0963 | |
| Gel dryer | Labconco | 4330150 | |
| Double emulsion autoradiography film | VWR | IB1651454 | |
| Film cassette | Fisher Scientific | FBAC-1417 | |
| Intensifying screen | Fisher Scientific | FBIS-1417 | |
| Plate sealing rubber roller | Sigma | R1275 |
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