使用新激酶蛋白的强生化方法在分子水平上进行表征
Summary
我们使用强大的生化方法对一种新的激酶蛋白进行了定性:用特殊抗体对不同细胞系和组织进行西带体分析,通过共免疫沉淀实验相互作用,由西方检测到的激酶活性使用磷特异性抗体和由 β+32P+ ATP 标记的斑点。
Abstract
广泛的全基因组测序已经确定了许多开放阅读框架(ORFs),提供了许多潜在的蛋白质。这些蛋白质可能对细胞有重要作用,并可能解开新的细胞过程。在蛋白质中,激酶是主要参与者,因为它们属于细胞信号通路,能够打开或关闭许多对细胞命运至关重要的过程,如细胞生长、分裂、分化、运动和死亡。
在这项研究中,我们重点研究了一种新的潜在激酶蛋白LIMK2-1。我们用特定的抗体证明了它的存在。我们使用共免疫沉淀实验评估其与上游调节蛋白的相互作用。共免疫沉淀是一种非常强大的技术,能够检测两个目标蛋白之间的相互作用。它也可以用来检测诱饵蛋白的新伙伴。诱饵蛋白可以通过设计到其序列的标签进行纯化,或者通过专门针对它的抗体进行纯化。然后,这些蛋白质复合物可以通过SDS-PAGE(硫酸二苯基苯甲酸钠聚丙烯酰胺凝胶)分离,并使用质谱法进行鉴定。免疫沉淀LIMK2-1也用于通过β+32P+ ATP标签在体外测试其激酶活性。这种成熟的测定可能使用许多不同的基质,诱饵的突变版本可用于评估特定残留物的作用。药理剂的作用也可以评估,因为这种技术是高度敏感和定量的。尽管如此,放射性处理需要特别小心。激酶活性也可以评估与特定抗体针对改性氨基酸的磷组。这些类型的抗体并非适用于所有磷改性残留物。
Introduction
几十年来,许多信号通路已被阐明,并表明它们参与关键的细胞过程,如细胞分裂、分化、运动、程序化细胞死亡、免疫和神经生物学。激酶在这些信号通路中起着重要作用,因为它们经常精细地调节其激活或失活,是响应外部刺激1,2,3的瞬态多功能复合物的一部分。激酶的突变和调节障碍经常导致人类疾病,因此在过去四十年中,它们已成为最重要的药物靶点之一。
在此背景下,能够检测激酶与其上游稳压器或下游基板的相互作用并识别新的合作伙伴非常重要。亲和纯化和免疫沉淀是分离蛋白质复合物5的非常强大的技术。诱饵蛋白或激酶可以标记与特定的肽序列允许使用商业珠子共价结合抗体针对肽。这种材料允许在实验6,7,8中具有很高的可重复性。内源性蛋白也可以使用直接靶饵蛋白的抗体进行免疫沉淀。抗体可能与蛋白A或蛋白GAagagarose珠子交联,或在添加赖沙之前用这些珠子孵育。莱沙缓冲液必须经过优化,使蛋白质溶解而不失去相互作用,并避免蛋白质降解。这种方法的一个主要缺点是相互作用在细胞赖沙时被检测到;因此,瞬态或弱相互作用,以及那些需要亚细胞上下文可能错过。其他技术可用于直接在细胞中工作,如接近结扎测定(PLA)9、体内交联辅助亲亲纯化(XAP)10、生物发光共振能量转移(BRET)或Fürster共振能量转移 (FRET)11,12。此外,免疫沉淀不适合确定结合的热力学常数,需要表面质原共振、等温分滴热量测定或微尺度热泳等物理技术13,14.
可使用多种技术评估激酶活性。在此,我们专注于磷特异性抗体和体外β=32P+ ATP(腺苷三磷酸)标签。磷特异性抗体针对蛋白质中特定残留物的磷酸盐修饰。它们可用于细胞裂解后西布洛特或ELISA(酶链接免疫吸附测定),用于免疫组织化学,以及使用流细胞学或免疫荧光的完整细胞。其缺点可能包括缺乏特异性,可以使用目标蛋白的突变版本进行评估,并且它们不能用于所有蛋白质。体外β=32P+ ATP标签是一种非常健壮、成熟和高度敏感的方法15。可使用免疫沉淀蛋白或重组蛋白,并测试不同的基质。药物的影响也可以评估,因为这种方法是定量的。其主要缺点是,与该方法相关的放射性需要谨慎处理。根据荧光或发光肽基质的测量,利用荧光/发光特性在磷酸化上改变的特性,也可以采用替代方法。这些方法还允许高通量,例如,在筛选可能成为目标激酶潜在抑制剂的分子时,这是必要的。事实上,激酶是制药公司追求的最大药物目标类别之一。
在这项研究中,我们重点研究了LIMK2-1蛋白(LIMK2-1代表Lin11,岛1,Mec3激酶等形2-1)。LIMK2激酶蛋白于1995年首次被描述17。通过替代拼接产生三种 LIMK2 等形:LIMK2a、LIMK2b 和 LIMK2-1。目前,LIMK2-1仅在一项研究18中描述了mRNA水平。在此,我们使用强大的生化方法在分子水平上描述这种潜在的新激酶蛋白。首先,我们证明了LIMK2-1确实是合成的。与LIMK2a和LIMK2b的两种对应物类似,它与上游激酶ROCK(Rho相关蛋白激酶)相互作用。我们显示LIMK2-1在Myelin基本蛋白(MBP)上有激酶活性,但在LIM激酶的规范基质cofilin上没有。
Protocol
Representative Results
Discussion
在这里,我们使用了强大的生化工具,在分子水平上描述一种新的蛋白质LIMK2-1,它被认为是基于其序列和同源性、LIMK2a和LIMK2b20的激酶。
首先,利用西布洛特与特定抗体的分析,在蛋白质水平上证明了LIMK2-1的存在。之后,我们评估了它与上游激酶ROCK1的相互作用,后者已知调节LIMK2a和LIMK2b,LIMK2-1的同源。最后,我们通过体外β32 P+ ATP标签和西布洛特在纤维素中使…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了癌症协会、L’L’协会神经纤维瘤病和雷克林豪森协会和瓦尔·卢瓦尔共和国中心的支持。非常感谢奥雷利·科松和德博拉·卡萨提供流式细胞学数据,感谢凯龙·希克曼-刘易斯对手稿进行彻底校对。
Materials
| Antibody anti-actin | Sigma-Aldrich | A1978 | for Western Blot |
| Antibody anti-c-Myc | Invitrogen | MA1-21316 | for Western Blot |
| Antibody anti-cofilin | Cell signaling Technology | 3312/5175 | for Western Blot |
| Antibody anti-GFP | Santa Cruz | sc-9996 | for Western Blot |
| Antibody anti-HA | Roche Applied Science | 11687423001 | for Western Blot |
| Antibody anti-phospho-cofilin | Cell signaling Technology | 3313 | for Western Blot |
| Antibody Anti-PP1i | Eurogentec | designed for this study | for Western Blot |
| Aprotinin | Euromedex | A-162B | for lysis buffer |
| ATP | Invitrogen | PV3227 | for g[32P] labeling |
| g[32P] ATP | Perkin Elmer | NEG502A | for g[32P] labeling |
| BES buffered saline | Sigma-Aldrich | 14280 | for transfection |
| b-glycerophosphate | Sigma-Aldrich | G9422 | for lysis and kinase buffer |
| b-mercaptoethanol | Sigma-Aldrich | M3148 | for Laemmli |
| BSA | Sigma-Aldrich | A3059 | for blocking buffer |
| Bromophenol Blue | Sigma-Aldrich | B0126 | for Laemmli |
| CaCl2 | Sigma-Aldrich | C3881 | for transfection |
| Centrifuge | Sigma | 111-541 | |
| Collagen R | Pan Biotech | P06-20166 | for transfection |
| Control siRNA | Ambion | AM4611 | for PP1i antibody specificity |
| Coomassie PageBlue Protein Staining Solution | Thermo-Fisher | 24620 | for gel staining |
| EDTA | Sigma-Aldrich | 3690 | for lysis buffer |
| Electrophoresis Unit | Biorad | Mini-Protean | for Western Blot |
| EZview Red anti-HA affinity gel | Sigma-Aldrich | E6779 | for immunoprecipitation |
| GeneSys software | Ozyme | for Western Blot acquisition | |
| GeneTolls software | Ozyme | for Western Blot quantification | |
| GFP-trap beads | Chromtek | for immunoprecipitation | |
| Glycine | Euromedex | 26-128-6405 | for transfer buffer |
| GST-cofilin | Upstate Cell signaling | 12-556 | for g[32P] labeling |
| Hamilton syringe 100 mL | Hamilton | 710 | to remove carefully supernatant from beads without aspirating them |
| HEPES | Sigma-Aldrich | H3375 | for kinase buffer |
| ImageQuant TL software | GE Healthcare | for radioactivity acquisition and quantification | |
| LIMK2 siRNA | Ambion | s8191 | for PP1i antibody specificity |
| Leupeptin | Sigma-Aldrich | SP-04-2217 | for lysis and kinase buffer |
| MBP | Upstate Cell signaling | 13-173 | for g[32P] labeling |
| MgCl2 | Sigma-Aldrich | M8266 | for kinase buffer |
| MnCl2 | Sigma-Aldrich | 244589 | for kinase buffer |
| NaCl | Euromedex | 1112 | for lysis and kinase buffer |
| NaF | Sigma-Aldrich | S-1504 | for lysis and kinase buffer |
| Okaidic acid | Euromedex | 0-2220 | for lysis buffer |
| PMSF | Sigma-Aldrich | 78830 | for lysis and kinase buffer |
| p-nitrophenylphosphate | Euromedex | 1026 | for lysis buffer |
| PVDF membrane Immobillon-P | Merck-Millipore | IPVH00010 pore size 0,45 mm | for Western Blot |
| Rotating wheel | Labinco | for bead incubation | |
| Safe lock eppendorf | Eppendorf | 0030120.086 | for kinase assay |
| SDS | Sigma-Aldrich | 5030 | for Laemmli and migration buffer |
| Sodium orthovanadate | LC Laboratories | S8507 | for lysis and kinase buffer |
| Sodium pyrophosphate | Fluka | 71501 | for lysis buffer |
| Super Signal West Dura | Protein Biology | 34075 | for Western Blot |
| Syngene Pxi | Ozyme | for Western Blot | |
| Tissue extracts |
Biochain |
P1234035 Brain P12345152 Lung P1234149 Liver P1234188 Pancreas P1234260 Testis |
for Western Blot analysis |
| Transfer Unit | Biorad | Mini-Trans-Blot | for Western Blot |
| Tris | Euromedex | 26-128-3094 B | for lysis buffer |
| Tween-20 | Sigma-Aldrich | P7949 | for blocking buffer |
| Typhoon FLA9500 | GE Healthcare | to read autoradiography | |
| Typhoon Trio | Amersham Bioscience | to read autoradiography | |
| Whatman paper | GE Healthcare | 3030-672 | for Western Blot |
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