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Summary
Abstract
Introduction
Protocol
Results
Discussion
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Acknowledgements
Materials
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癌症研究

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

Published: July 17, 2019
doi:
10.3791/59795
, , , , ,
1The Laboratory of Cancer Signaling, Division of Cellular and Molecular Research,National Cancer Centre Singapore, 2The Cancer and Stem Cell Program,Duke-NUS Medical School

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1. 用于测量 RAF 突变体催化活性的 Vitro 激酶测定 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)。 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;(…

Representative Results

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…

Discussion

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,?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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)。

Materials

anti-phosphoERK1/2 Cell Signaling Technologies 4370
anti-phosphoMEK1/2 Cell Signaling Technologies 9154
anti-ERK1/2 AB clonal A0229
anti-MEK1/2 Cell Signaling Technologies 9124
anti-FLAG(mouse) Sigma-Aldrich F3165
anti-HA Novus Biologicals MAB6875
anti-FLAG(Rabbit) Cell Signaling Technologies 14793
anti-β-actin Sigma-Aldrich A2228
anti-FLAG beads(M2) Sigma-Aldrich A4596
HRP-conjugated anti-mouse IgG Jackson Laboratories 115-035-003
HRP-conjugated anti-Rabbit IgG Jackson Laboratories 111-035-144
pcDNA3.1(+) In vitrogen V79020
Gibson Assembly Cloning  Kit New England Biolabs E5510
T4 DNA ligase New England Biolabs M0202
lipofectamine 2000 Invitrogen 11668019
Fugene 6 Roche 11 814 443 001
DMEM w/o phenol red Invitrogen 21063-029
D-luciferin  GoldBio LUCK-100
6xhis-tagged MEK1 (K97A)  prepared in our previous studies N.A. Reference 15.
GloMax-Multi Detection System. Promega E7041
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References

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Tags

Keywords: RAF KinaseDisease-related MutantsCharacterizationCatalytic ActivityFlag-taggedGlutathione S-transferase293T CellsLysis BufferAnti-FLAG Affinity BeadsKinase ReactionSDS-PAGEImmunoblot
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Yap, J., Yuan, J., Tee, Z. H., Huang, X., Ng, W. H., Hu, J. Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods. J. Vis. Exp. (149), e59795, doi:10.3791/59795 (2019).
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