Name: identification of kinase-substrate pairs using high throughput screening
Uploaded: 2015-08-29T14:00:00.000+00:00
Duration: 11 min 13 s
Description: Watch this Scientific Journal Video about Identification of Kinase-substrate Pairs Using High Throughput Screening at JoVE.com

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。

1.准备试剂，板，和细胞<ol><li>制成500 ml的裂解缓冲液:25毫摩尔Tris pH值7.5，150mM的氯化钠，50mM的氟化钠，0.5mM EDTA的pH为8.0，0.5％的TritonX-100，5mM的β-内甘油，5％甘油。保存在4℃。使用，添加1mM的二硫苏糖醇（DTT），1mM的苯甲基磺酰氟（PMSF），和1mM钒酸钠之前立即。在该步骤之后，PMSF中不需要任何漂洗缓冲液中。 </li><li>使将20ml 10×激酶缓冲液:200毫摩尔Tris pH为7.5，50mM的β-内甘油（FW 216），2mM的钒酸钠。分装在1ml试管并储存在-20℃。使用，加上5 mm DTT前夕。 </li><li>使20ml中10倍的M-ATP的缓冲液:300μM三磷酸腺苷（ATP）的66mM的MgCl 2，33毫摩尔的MnCl 2，分装在1ml试管并储存在-20℃。 </li><li>使100个毫升2×十二烷基硫酸钠（SDS）裂解缓冲液:1.5克的TRIS碱，将20ml甘油，加入30ml 的 H 2 O.溶解并调整pH至6.8，用HCl。添加40毫升10％SDS中，音量调节至100毫升。加入25毫克的溴酚蓝。 </li><li>现货4微升25毫微克/微升哺乳动物表达质粒套96孔板和标签板相应的编码每一个GST激酶14家 。密封和冷冻板在-20℃下直到使用。 </li><li> 1-2天前，转染，培养HEK293T细胞中完整的Dulbecco改良的Eagle培养基（DMEM）+ 10％胎牛血清（FBS）和抗生素在37℃培养箱中补充有二氧化碳 （最终5％）。通道与胰蛋白酶和不允许的股票成为&gt; 80％汇合膨胀的过程中。至少6×10 7个细胞的所需屏幕（约3×15厘米，在80％汇合HEK293T细胞的碗碟）。 </li></ol> 2.转染注: 图 1所示为整个协议的流程图。 <ol><li>如果包含激酶质粒板已被冻结，在室温下解冻一次离心在1900×g离心3分钟以收集任何湿气在孔的底部。 </li><li>拌8.6 ml的低血清培养基（例如，OPTI-MEM）与312.7微升基于脂质的转染试剂的。让我们坐5分钟。 </li><li>加10微升减少血清的培养基，每孔使用自动化液体分配器装配有小体积盒。 </li><li>添加每降低血清培养基/转染试剂混合物从步骤2.2的井10微升用自动液体分配器装配有小体积盒。让我们坐20〜45分钟。 </li><li>重悬293T细胞在在80ml完全DMEM的7.5×10 5个细胞/ ml。加入100微升细胞悬浮液（即7.5×10 4个细胞）的每使用自动化液体分配器装有标准量盒式良好。 </li><li>检查井显微镜均匀细胞分布下并返回到培养箱24小时。 </li></ol> 3. GST激酶下拉<ol><li>让˚FRESH:4mM的通过混合60微升的0.2M钒酸钠与540微升H 2 O过钒酸盐溶液在第二管混合2.7微升过氧化氢的30％和1.4毫升的PBS。添加两种方法结合，让坐在使用前15分钟。 </li><li>使用多道移液器（因为它在井产生太多的湍流不使用自动化液体分配器）分配2微升0.25M的氯化钙 2在各孔中，接着2.5微升在步骤3.1中制备的过钒酸盐的。孵育各板在37℃下进行10分钟，然后放置在冰上。 </li><li>通过添加DTT，PMSF，和钒酸钠如第1（试剂的配制）所示制备的35ml裂解缓冲液。 </li><li>保持板置于冰上，利用真空抽吸每孔删除网上平台。立即用自动液体分配器装有标准盒式添加50μl/孔的冰冷的裂解缓冲液。让我们坐30分钟，在冰上裂解（可选:可以在这里如果有必要通过密封高原停止e和储存在-80℃。要继续，解冻板冰）。 </li><li>旋板在1900×g离心3分钟，在4℃。 </li><li>使用多道移液器向各孔刮去细胞和转移所有内容以适当标记的V型底96孔板中。旋板在1900 xg离心在4℃下10分钟。 </li><li>期间的旋转，填充谷胱甘肽包被的板（每个96孔板）用100μl/孔的冰冷裂解缓冲液（无PMSF），为漂洗。置于冰上板。 </li><li>从离心机中取出V形底平板。一次一个，反转谷胱甘肽板在水槽上方，以摇出的纸巾裂解液和印迹。通过倾斜板，并使用多道移液器，小心不要在底部打扰颗粒移植自V-底板的谷胱甘肽板裂解液。盖板和置于冰上至少2小时进行绑定。 </li><li>接近2小时的结合步骤的结束时，准备一个放射性工作站，确保吨他必要的安全防范措施到位放射性工作。设置杂交炉至30℃。 </li><li>如在第1节PMSF表明不需要在该阶段制得200毫升裂解缓冲液加入DTT和钒酸钠。 </li><li>反转的谷胱甘肽板在水槽上方，以摇出裂解液涂抹在纸巾上。冲洗孔3倍用100μl裂解缓冲液（无PMSF）。不要让井坐干 - 让他们在漂洗，直到准备进行。 </li><li>通过稀释10倍的库存和添加DTT作为用自动液体分配器装有标准体积盒在第1指示的冲洗板一次用50μl的1×KB的制备55毫升1×激酶缓冲液（KB）。留1个KB的井，直到溶液中的准备: <ol><li>通过添加500至530微克的兴趣将基片，500微克的髓鞘碱性蛋白（MBP），2.65毫升10×KB，13.25微升的1M DTT和H 2 O的高达15.9 ml的制备溶液A。 </li></ol></LI&gt; <li>一次一个，翻转板在水槽上方，除去1个KB冲洗，涂抹在纸巾上，并立即使用自动液体分配器配有体积小纸盒加30微升溶液A。置于冰上板。 </li><li>制备溶液B在放射性工作区域通过加入2.5 ml的10倍的M-ATP，500微居里的γ-32 p ATP和H 2 O的至10ml。 <ol><li>加入20微升溶液B每使用中继器吸管，它有助于混合，由于喷射力很好。盖上盖子进行培养在30℃杂交炉30分钟。 </li></ol></li><li> 30分钟后，回转板，以冰。加入50微升2×SDS裂解缓冲液，使用多道移液器每个孔中。可以进行在这个点到下一个步骤，或密封用铝箔和存储所述板在-20℃直至方便。 </li></ol> 4.运行，染色，干燥凝胶注:所有工作都应该在一个地区DESIGNA进行泰德放射性。 <ol><li>打开杂交炉和设定为85℃。解冻板在室温下。一旦烘箱达到温度​​，传热板到烤箱并孵育10分钟以变性样品。 </li><li>负载26孔预制胶与15微升使用多道移液器每一个反应，以填补多口井一次。必须注意，所有的技巧配合以及相对应先加样品。运行凝胶在150五，不要让示踪染料（蓝线）跑出凝胶的底部，因为这包含了未结合的ATP。 </li><li>拆除凝胶和切断使用手术刀或直边，因为它会曝光过度，影片的未结合的ATP（蓝线）。将凝胶在标签的容器和盖用考马斯染色15分钟。 </li><li>取出考马斯染色，简单地用清水冲洗凝胶，并添加脱色的解决方案。脱色的凝胶直至蛋白质都清晰可见。 A波段的MBP和基材带应对于每个样品可见。 </li><li>以干燥的凝胶: <ol><li>切滤波器一张大纸，并把它放在烘干机。 </li><li>湿蒸馏水玻璃纸片，直到它是光滑，无皱，并将其放置在纸张的顶端。 </li><li>躺在玻璃纸片材的顶部的凝胶，使得记凝胶的量级。湿的第二玻璃纸片和放在凝胶的顶部。 </li><li>铺开所有的泡沫（一板的密封辊可以很好地用于本）的一个很好的均匀的表面。关闭挡板，打开真空，并在80℃干燥该凝胶3小时。 </li></ol></li><li>一旦凝胶干燥，用丝网加紧信号，将其暴露在XAR胶片。用保鲜保鲜膜或塑料袋磁带，并用胶带密封，保持了霜冻。存储盒在-80℃下过夜。 </li></ol> 5.制定XAR电影<ol><li>翌日，从冷冻库取出暗盒，并让在室温下解冻。发展中的膜使用薄膜处理器根据制造商的说明暗室。 注意:检查薄膜激酶底物对的证据。阿秒更长的曝光也可以是有用的检测更弱的磷酸化事件。 </li></ol>

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The authors have nothing to disclose.

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...

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。

<table><tbody><tr><td>Lysis buffer</td><td>Made in house</td><td></td><td>See Protocol step 1.1</td></tr><tr><td>10x kinase buffer</td><td>Made in house</td><td></td><td>See Protocol step 1.2</td></tr><tr><td>10x M-ATP</td><td>Made in house</td><td></td><td>See Protocol step 1.3</td></tr><tr><td>Human kinase plasmids</td><td>Orfeome, Invitrogen, Origene</td><td></td><td>GST-tagged in house</td></tr><tr><td>96 well plates</td><td>Fisher Scientific</td><td>CS003595</td><td></td></tr><tr><td>293T cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>DMEM</td><td>Fisher Scientific</td><td>SH3002201</td><td>supplement with 100&amp;nbsp;U/ml penicillin, 100&amp;nbsp;&amp;#956;g/ml streptomycin, 10% fetal calf serum.</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Sanyo</td><td>MCO-17AIC</td><td></td></tr><tr><td>15 cm cell culture dishes</td><td>Fisher Scientific</td><td>877224</td><td></td></tr><tr><td>Reduced serum medium</td><td>Invitrogen</td><td>22600-050</td><td></td></tr><tr><td>Lipid-based transfection reagent</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>Automated liquid dispenser</td><td>Thermo Scientific</td><td>5840300</td><td></td></tr><tr><td>Small cassette attachment</td><td>Thermo Scientific</td><td>24073295</td><td></td></tr><tr><td>Standard cassette attachment</td><td>Thermo Scientific</td><td>14072670</td><td></td></tr><tr><td>4 mM pervanadate</td><td>Made in house</td><td></td><td>See Protocol step 3.1</td></tr><tr><td>0.25 M CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Made in house</td><td></td><td></td></tr><tr><td>Multichannel pipette (20-200 &amp;#956;l)</td><td>Labnet</td><td>p4812-200</td><td></td></tr><tr><td>Multichannel pipette (1-10 &amp;#956;l)</td><td>Thermo Scientific</td><td>4661040</td><td></td></tr><tr><td>V-bottom 6-well plates</td><td>Evergreen Scientific</td><td>290-8116-01V</td><td></td></tr><tr><td>Glutathione coated 96-well plates</td><td>Fisher Scientific</td><td>PI-15240</td><td></td></tr><tr><td>Hybridization oven</td><td>Biostad</td><td>350355</td><td></td></tr><tr><td>GST tagged substrate</td><td>Made in house</td><td></td><td></td></tr><tr><td>Myelin Basic Protein (MBP)</td><td>Sigma</td><td>M1891</td><td></td></tr><tr><td>Repeater pipette (1 ml)</td><td>Eppendorf</td><td>22266209</td><td></td></tr><tr><td>&lt;sup&gt;32&lt;/sup&gt;P gamma-ATP</td><td>Perkin Elmer</td><td>BLU502Z500UC</td><td></td></tr><tr><td>2x SDS lysis buffer (100&amp;nbsp;ml)</td><td>Made in house</td><td></td><td>See Protocol step 1.4</td></tr><tr><td>26-well precast TGX gels</td><td>BioRad</td><td>567-1045</td><td>gel percentage required is dependent on the molecular weight of the substrate of interest</td></tr><tr><td>Coomassie stain</td><td>Made in house</td><td></td><td>0.1% Coomassie R250, 10% acetic acid, 40% methanol</td></tr><tr><td>Coomassie destain</td><td>Made in house</td><td></td><td>10% acetic acid, 20% methanol</td></tr><tr><td>Labeled gel containers</td><td>Made in house</td><td></td><td>Used plastic lids from empty tip boxes, just big enough to contain one gel</td></tr><tr><td>Whatman filter paper</td><td>Fisher Scientific</td><td>57144</td><td></td></tr><tr><td>Cellophane sheets (2)</td><td>BioRad</td><td>165-0963</td><td></td></tr><tr><td>Gel dryer</td><td>Labconco</td><td>4330150</td><td></td></tr><tr><td>Double emulsion autoradiography film</td><td>VWR</td><td>IB1651454</td><td></td></tr><tr><td>Film cassette</td><td>Fisher Scientific</td><td>FBAC-1417</td><td></td></tr><tr><td>Intensifying screen</td><td>Fisher Scientific</td><td>FBIS-1417</td><td></td></tr><tr><td>Plate sealing rubber roller</td><td>Sigma</td><td>R1275</td><td></td></tr></tbody></table>

identification of kinase-substrate pairs using high throughput screening

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。

来自屏幕代表性的结果示于图2。180激酶使用来自CRTC2以及经典激酶测定衬底髓鞘碱性蛋白（MBP）相应于氨基酸268-283一个GST-标记的肽底物进行筛选。只有两个激酶MARK2和高度相关激酶磷酸化MARK3的CRTC2肽。 MBP被包括在所有测定中的内部对照，因为它包含了许多可磷酸化的残基，并运行在18 kDa的，朝向所述凝胶的底部。这允许特异性的解释:有些激酶将有力磷酸化底物和MBP。?...

Watch this Scientific Journal Video about Identification of Kinase-substrate Pairs Using High Throughput Screening at JoVE.com

Identification of Kinase-substrate Pairs Using High Throughput Screening

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 ...

identification-kinase-substrate-pairs-using-high-throughput

Research

JoVE Journal

Biology

8.1K 次观看.  Children's Hospital of Eastern Ontario Research Institute. 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.

视频: Identification of Kinase-substrate Pairs Using High Throughput Screening

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 ...

科研

生物化学

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

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; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

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

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

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; ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

通过高通量抑制剂筛选鉴定调节胚胎干细胞多能性的激酶的简单方法

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

发育生物学

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 ...

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 ...

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 ...

生物学

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

基于生物传感器的高通量生物跨越和生物信息学分析战略，用于全球药物-蛋白质相互作用验证

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

生物工程

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向整流钾通道的小分子调节剂的高通量筛选

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...