Name: characterize disease-related mutants of raf family kinases by using a set of practical and feasible methods
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作者感谢毛细胞白血病奖学金对袁继明的支持。这项工作得到了亚洲基金癌症研究(AFCR2017/2019-JH)、杜克-新加坡国立大学Khoo桥资助奖(杜克-NUS-KBrFA/2018/0014)、NCCRF桥接赠款(NCCRF-YR2018-JUL-BG4)、NCCRF试点赠款(NCCRF-YR2017-JUL-PG3)和SHF学术资助。研究补助金(AM/TP011/2018)。

1. 用于测量 RAF 突变体催化活性的 Vitro 激酶测定
<ol><li>使用吉布森组装或传统的分子克隆方法,在C-总站用FLAG(DYKDDDDK)标记构建用RAF突变体编码的载体(图1A)。
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<li>通过 PCR 将 FLAG 标记和突变引入 RAF 编码序列,然后使用吉布森组件或 T4 DNA 结扎并遵循制造商的协议,将整个序列插入 pCDNA3.1(+) 载体中。对PCR反应使用以下条件:(1) 95 °C,2分钟;(2) 95 °C, 30 s;(3) 59 °C, 30 s;(...

<ol>
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在本文中,我们提出了三种描述与疾病相关的RAF突变体的特征的方法,包括体外激酶测定、RAF共激活测定和免费裂化荧光酶测定。由于RAF激酶具有催化活性和铀活性,各种RAF突变体可以通过两个不同的机制13、14、16、17激活下游信号。18.构成活性RAF突变体直接磷化下游效应器MEK,?...

RAF家族激酶是RAS/RAF/MEK/ERK信号级联的关键组成部分,它传输来自RAS的信号来激活线粒体激活蛋白激酶(MEK)1、2、3、4。这个家族激酶在细胞生长、存活和分化中起着至关重要的作用,它们的改变诱发了许多疾病,特别是癌症5,6,7,8。最近,基因组测序已经发现许多与疾病有关的RAF突变体在RAS/RAF/MEK/ERK级联9、10、11的信号传输中表现出不同的特性。仔细描述RAF突变体将有助于我们了解RAF突变体如何改变RAS/RAF/MEK/ERK级联的信号输出的分子机制,最终选择适当的方法来治疗各种RAF突变体驱动的疾病。
皇家空军家族激酶包括三个成员,CRAF,BRAF和ARAF,它们具有相似的分子结构,但激活下游信号1,2,3,4的能力不同。在这些参数中,BRAF 因其构成性磷酸化 NtA (N-t erminal acidic) 主题12、13、14 而 ARAF 具有最低的活性活动产生于其非规范的APE主题15。这可能解释不同突变频率的皇家空军参数在疾病:BRAF>CRAF>ARAF。此外,在同一个 RAF 参数中,不同站点的突变可能会以不同的方式触发下游信号,这为 RAF 家族激酶的调节增加了另一层复杂性。最近的研究表明,所有皇家空军激酶都有催化和阿尔波斯特活动13,14,16,17,18。组织活性RAF突变体通过磷化MEK直接打开下游信号,而激酶死RAF突变体可以通过侧对侧二聚变并激活MEK-ERK信号16转动其野生型对等体 ,19,20.二角亲和力/稳定性是一个关键参数,它不仅决定激酶死RAF突变体的碱性活性,而且影响构成活性RAF突变体的催化活性15,2122.具有高二聚体亲和力/稳定性的激酶死原生突变体可以直接将内源性野生型RAF转动15,而那些具有中间二聚体亲和力/稳定性的突变体需要主动Ras或高度的野生型RAF分子功能13,15,20,21,23。同样,构成活性RAF突变体以二分位依赖方式磷化MEK,而那些具有低二分位亲和力/稳定性的突变体在免疫沉淀后失去其在体外催化活性,而免疫沉淀打破了弱的RAF二聚体15, 21,22.二聚体亲和力/稳定性也决定了RAF突变体对其抑制剂的敏感性,并与RAF抑制剂24的抗性呈正相关。因此,为了描述与疾病相关的RAF突变体,有必要测量其催化和碱化活性,以及二角体亲和力/稳定性。
近年来,我们的实验室和其他实验室已经开发出各种方法来表征皇家空军家族激酶及其突变体。根据我们的实验室和其他年的经验,我们认为以下三种检测在定义与疾病相关的RAF突变体方面具有优势:(1)易于检测组织催化活性的体外激酶测定活性RAF突变体15;(2)RAF联合激活测定,这是一个可靠和方便的方法,以测量激酶死亡的RAF突变体13,15,21,22,23的活性。25;(3) 与传统的共免疫沉淀测定相比,在测量RAF突变体的相对二聚位亲和/稳定性方面具有更高灵敏度的免费裂化荧光酶测定,并且能够在没有先进设备的情况下进行。与定量分析方法如SPR(S尿面P拉斯蒙Resonance)分析15,22。结合这三种检测,我们可以很容易地了解与疾病相关的RAF突变体如何改变下游信号,从而利用适当的治疗策略来治疗由此RAF突变引起的疾病。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-phosphoERK1/2</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">4370</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-phosphoMEK1/2</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">9154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-ERK1/2</td>
			<td style="width:197px;">AB clonal</td>
			<td style="width:104px;">A0229</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-MEK1/2</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">9124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-FLAG(mouse)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:104px;">F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-HA</td>
			<td style="width:197px;">Novus Biologicals</td>
			<td style="width:104px;">MAB6875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-FLAG(Rabbit)</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">14793</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-&#946;-actin</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:104px;">A2228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-FLAG beads(M2)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:104px;">A4596</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">HRP-conjugated anti-mouse IgG</td>
			<td style="width:197px;">Jackson Laboratories</td>
			<td style="width:104px;">115-035-003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">HRP-conjugated anti-Rabbit IgG</td>
			<td style="width:197px;">Jackson Laboratories</td>
			<td style="width:104px;">111-035-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">pcDNA3.1(+)</td>
			<td style="width:197px;">In vitrogen</td>
			<td style="width:104px;">V79020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Gibson Assembly Cloning&#160; Kit</td>
			<td style="width:197px;">New England Biolabs</td>
			<td style="width:104px;">E5510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">T4 DNA ligase</td>
			<td style="width:197px;">New England Biolabs</td>
			<td style="width:104px;">M0202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">lipofectamine 2000</td>
			<td style="width:197px;">Invitrogen</td>
			<td align="right" style="width:104px;">11668019</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Fugene 6</td>
			<td style="width:197px;">Roche</td>
			<td style="width:104px;">11 814 443 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">DMEM w/o phenol red</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:104px;">21063-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-luciferin&#160;</td>
			<td style="width:197px;">GoldBio</td>
			<td style="width:104px;">LUCK-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">6xhis-tagged MEK1 (K97A)</td>
			<td style="width:197px;">&#160;prepared in our previous studies</td>
			<td style="width:104px;">N.A.</td>
			<td>Reference 15.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">GloMax-Multi Detection System.</td>
			<td style="width:197px;">Promega</td>
			<td style="width:104px;">E7041</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterize disease-related mutants of raf family kinases by using a set of practical and feasible methods

迅速加速的纤维肉瘤(RAF)家族激酶在细胞生物学中起着核心作用,其功能障碍导致癌症和发育障碍。与疾病相关的RAF突变体的特征将帮助我们选择适当的治疗策略来治疗这些疾病。最近的研究表明,RAF家族激酶具有催化和脱硫活性,受二聚体化的严格调节。在这里,我们构建了一套实用可行的方法,以确定RAF家族激酶及其突变体的催化和碱性活动以及相对二聚体亲和力/稳定性。首先,我们修正了经典的体外激酶测定,通过降低缓冲液中的洗涤剂浓度,采用温和的快速洗涤程序,并采用谷胱甘肽S-转移酶(GST)融合,以防止RAF二聚物在净化。这使我们能够适当地测量组织活性RAF突变体的催化活性。其次,我们开发了一种新型的RAF共激活测定法,通过使用N端截断的RAF蛋白来评估激酶死原体突变体的机分活性,消除了当前协议中活性Ras的需求,从而实现了更高的灵敏度。最后,我们生成了独特的互补裂化荧光素酶测定法,定量测量了各种RAF突变体的相对二聚体亲和力/稳定性,与传统共免疫沉淀测定相比,它更可靠、更灵敏。总之,这些方法具有以下优点:(1) 用户友好;(二)无先进设备,能够有效进行;(3) 性价比高;(4) 高度敏感且可重复。

皇家空军家族激酶具有催化和异体活动,这使得其与疾病有关的突变体能够通过不同的机制打开下游信号13、14、16、17 ,18.构成活性的RAF突变体直接磷化其基质,而激酶死RAF突变体通过转活野生型对等体实现其功能。如图1B所示,两种构成活性 RA...

Watch this Scientific Journal Video about Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods at JoVE.com

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

在本文中,我们提出了一套实用可行的方法,用于描述RAF家族激酶与疾病相关的突变体,包括体外激酶测定、RAF共活化测定和补充裂化荧光酶测定。

使用一套实用可行的方法,描述英国皇家空军家族激酶与疾病相关的突变体

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

这种方法有助于有效描述与疾病相关的RAF激酶方法。该技术高度敏感且可重复性，经济高效，用户友好，无需任何先进设备。为了测量RAF突变体的催化活性，在根据标准协议将整个序列插入适当的载体之前，通过PCR将标志技术突变引入RAF编码序列。将谷胱甘肽插入到RAF突变编码序列上游的转移酶编码序列，以同样的方式生成编码谷胱甘肽为转移酶融合RAF突变体的载体。根据标准协议，Transvect 80%至 90% 汇合 293T 细胞培养与向量编码标志标记 RAF 激酶或其突变体。经过24小时的培养，更换每种文化中的媒介。培养48小时后，在冰上每一个井中加入400微升的解液缓冲液，并辅以蛋白酶和磷酸酶抑制剂。5 至 10 分钟后，将细胞乳酸转移到 1.5 毫升管中，用于离心沉淀细胞碎屑。将每个样品的300微升转移到单独的1.5毫升微离心管中。每个样品留出40微升用于下游批发莱酸盐化石外细胞信号调节激酶一和二表达和活动氨基印迹分析。在每个管中加入20微升的抗旗亲和力珠，在4摄氏度的冷室中旋转一小时。在孵育结束时，快速轻轻清洗抗旗珠，在冷室用解液缓冲液轻轻清洗一次，用激酶反应缓冲液轻轻清洗一次，然后加入20微升激酶反应混合物。具有低Dima亲和力的图形突变体可能会因为Dima分离而失去体外催化活性。因此，快速和温和的洗涤是至关重要的。在室温下10分钟后每隔一分钟翻转一次，用5个XSDS样品缓冲液的5微升停止激酶反应，每个样品在90摄氏度下煮沸样品5分钟。在孵化结束时，使用0.1%SDS在9至12%聚丙烯酰胺电泳凝胶上运行样品，然后将蛋白质转移到硝基纤维素膜上，以便通过免疫印迹检测每个样品中磷质三粒细胞活性蛋白激酶和RAF突变体的水平。要评估激酶死亡皇家空军突变体的蛋白质组活性，构建编码RAF接收器或激酶死RAF活化剂的载体。Transvect 293 T 细胞具有两个载体编码 RAF 接收器和激酶死 RAF 活化剂， 或单个向量编码的蛋白质之一， 如证明.如所示，在培养48小时后收获整个细胞的乳酸，并在室温下将干净的全细胞乳酸与5个XSDS样品缓冲液快速混合，在90摄氏度下将乳酸盐煮沸5分钟。然后运行煮沸的整细胞解酶样品在9至12%聚丙烯酰胺电泳凝胶为0.1%SDS和转移蛋白质到硝基纤维素膜检测磷外细胞信号调节激酶一和二的水平，并控制蛋白质通过免疫印迹，如证明。测量RAF突变体的相对暗淡亲和力和或稳定性，构建编码旗技术草案突变体的载体，融合到萤火虫荧光酶的端子或萤火虫荧光酶的C术语。Transvect 293 T 细胞与一对载体编码不同和术语的萤火虫荧光酶 RAF 突变体和 C 头科一个萤火虫荧光酶 RAF 突变体如证明。24小时后转盘两次10至第五293T细胞事务每井成水晶黑色图像板在无色介质。转液后48小时将荧光素加入293T细胞转维，孵育30分钟。在测量多检测系统上的荧光素酶信号之前。在测量结束时，对超吸量进行吸气，并用 lysas 缓冲液对 293 个 T 细胞进行飞毛水，如所证明的。使用 0.1% SDS 在 9 至 12% 聚丙烯酰胺电泳凝胶上运行整个细胞解质样品。检测萤火虫荧光酶RAF突变体的结束终点的表达水平，或萤火虫荧光酶RAF突变体的C终点通过反旗氨基印迹的表达水平，如所证明的。然后根据萤火虫荧光酶末和萤火虫荧光酶RAF突变体结束终点的表达水平，使293个T细胞转越子的荧光酶信号正常化。体外激酶 asa 可用于有效探测一些有组织活性的 R-spine 突变体，但不包括具有融合 C-spine 的激酶死亡突变体。然而，具有弱二暗亲和力或稳定性的组成活性RAF突变体的催化活性，可能无法通过使用这种 ASA 来探测，因为其二氧化二暗体在纯化过程中被打破。为了避免RAF突变体失去催化活性，具有弱的二丁体亲和力或稳定性，这些突变体可以与谷胱甘肽转移酶融合，以挽救突变体的催化活性。皇家空军共同激活 ASA 可用于评估激酶死亡 RAF 突变体的同性活性。如图示激酶死突变体与酸性端端质图案和C脊柱融合，功能作为同酸性活化剂触发突变体与非磷相对酸性终端图案的催化活性，当共同表达在293T细胞。此外，免费分裂荧光素酶SA可用于测量RAF家族激酶及其突变体的相对暗淡亲和力或稳定性。使用低浓度洗涤剂的 lysas 缓冲液，并在样品纯化过程中快速温和的洗涤程序对于成功的体外激酶 SA 至关重要。这项技术将帮助研究人员准确确定疾病如何传递RAF突变体，激活下游途径，促进疾病治疗的适当治疗策略的选择。

Research

JoVE Journal

Cancer Research

A Mouse Model of Fatigue Induced by Peripheral Irradiation

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

科研

癌症研究

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

应用肺转移法研究骨肉瘤转移性肺定植的实用思考

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

PET 与 MRI 引导下微器对大鼠胶质瘤模型的照射

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Detection of Protease Activity by Fluorescent Peptide Zymography

荧光肽酶谱法检测蛋白酶活性

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

68型 Ga-DOTATATE 神经内分泌肿瘤的日常临床应用指南

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

In Vitro Assay to Study Tumor-macrophage Interaction

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

通过测量活细胞中的 eIF4E-eIF4G 交互来监控 eIF4F 装配

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

利用计算机图像分析提高4T1乳腺癌模型中肺转移的定量

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

RNA 测序的三种微分表达分析方法：利马、边缘、DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration ...