用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性
Summary
本研究介绍了一种简单的方法检测内源性水平的 Rab10 磷酸化富亮氨酸重复激酶2。
Abstract
富含亮氨酸重复激酶 2 (LRRK2) 的突变被证实与家族性帕金森病 (平板) 有关。由于 LRRK2 激酶活性的异常活化已经牵连到 PD 的发病机制中, 因此建立一种评价 LRRK2 激酶活性的方法是十分必要的。最近的研究表明, LRRK2 对酶家族的成员, 包括 Rab10, 在生理条件下。虽然在培养细胞中 LRRK2 内源性 Rab10 的磷酸化可以通过质谱法检测, 但由于目前可用的磷酸化特异抗体的敏感性差, 很难通过免疫检测Rab10在这里, 我们描述了一个简单的方法, 检测 Rab10 磷酸化的 LRRK2 基于免疫利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 结合磷酸盐结合标签 (P 标签), 这为N-(5-(2-aminoethylcarbamoyl) 吡啶-2-ylmetyl)-n,n “,n”-三 (吡啶-2-基-甲基)-13-diaminopropan-2-ol。本议定书不仅提供了利用 P 标记的方法的一个例子, 而且还能够评估突变以及抑制剂治疗/管理或任何其他因素如何改变 LRRK2 在细胞和组织中的下游信号.
Introduction
PD 是最常见的神经退行性疾病之一, 主要影响多巴胺能神经元在中脑, 导致功能障碍的老年人的马达系统的1。虽然大多数病人是以零星的方式发展 PD, 但仍有家庭继承这种疾病。一些基因的突变被发现与平板的2相联系。平板的致病基因之一是 LRRK2, 其中八义突变 (N1437H, R1441C/G/H/S, Y1699C, G2019S, I2020T) 链接到一个主导的遗传的平板被称为 PARK8 迄今已被报告3,4,5。数个全基因组的联合研究 (GWAS) 的零星 pd 患者也确定了 LRRK2 位点的基因组变异作为帕金森病的危险因素, 这表明 LRRK2 的功能异常是神经的常见原因。和 PARK8 平板显示器6,7,8。
LRRK2 是一种大蛋白质 (2527 氨基酸), 由富含亮氨酸的重复域、GTP 复合蛋白 (roc) 域的结合 Ras、roc (林) 域 C 端、丝氨酸/苏氨酸蛋白激酶域和 WD40 重复域9组成。八平板的突变定位在这些功能领域;N1437H 和 R1441C/G/H/S 在 ROC 域, Y1699C 在林域, G2019S 和 I2020T 在激酶领域。自 G2019S 突变, 这是最常见的突变在 PD 患者10,11,12, 增加了激酶活性的 LRRK2 2-3 倍在体外13, 这是假设LRRK2 基质磷酸化的异常增加对神经元有毒性。然而, 由于缺乏对患者的样本进行评价的方法, 因此无法研究在家族性/偶发性 PD 患者中是否改变了生理相关 LRRK2 基质的磷酸化。
蛋白质磷酸化通常是通过免疫或酶联免疫吸附试验 (ELISA), 使用抗体专门识别磷酸化状态的蛋白质或质谱分析。然而, 前者的策略有时不能应用, 因为在创建磷酸化特异抗体的困难。代谢标记的细胞与放射性磷酸盐是另一个选择, 以检查的生理水平磷酸化特定抗体是不容易获得。然而, 它需要大量的放射性材料, 因此涉及一些专用设备, 用于防护14。与这些免疫方法相比, 质谱分析更加灵敏, 在蛋白质磷酸化分析中得到了广泛的应用。然而, 样品准备是费时的, 并且昂贵的仪器是需要的分析。
酶家族的一个子集, 包括 Rab10 和 Rab8, 最近被报告为 LRRK2 的直接生理基质, 根据 large-scale phosphoproteomic 分析结果15。然后我们证明, Rab10 磷酸化是增加了小鼠胚胎成纤维细胞的平板的突变和在敲鼠标的肺部16。在这份报告中, 我们选择采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (sds 页) 为基础的方法, 其中 p 标记分子 co-polymerized 到 sds 页凝胶 (p 标签 sds 页), 以检测内源水平的 Rab10 磷酸化,因为对磷酸化 Rab10 特异性强的抗体仍然缺乏。我们没有检测到内源性 Rab8 的磷酸化, 由于目前可用的抗体对总 Rab8 的选择性较差。因此, 我们决定专注于 Rab10 磷酸化。LRRK2 对 Rab10 在 Thr73 位于高度保守的 “开关 II” 区域的中间。高保护的磷酸化点之间的蛋白质可能是一个原因, phosphospecific 抗体识别不同的蛋白质是很难作出的。
Rab8A 的磷酸化 LRRK2 抑制 Rabin8 的结合, 一种鸟嘌呤核苷酸交换因子 (GEF), 它通过与 GTP15交换绑定的 GDP 来激活 Rab8A。LRRK2 的磷酸化 Rab10 和 Rab8A 也抑制了国内生产总值-离解抑制剂 (GDIs) 的结合, 这对于通过从膜中提取国产总值束缚的蛋白从15中活化蛋白质是必不可少的。总的来说, 它是假设, LRRK2 的蛋白质磷酸化防止他们的活化, 虽然确切的分子机制和生理后果的磷酸化仍然不清楚。
P 标记 SDS-页是由木et al.在2006年发明的: 在这种方法中, 丙烯酰胺与 P 标记共价结合, 一个分子捕获高亲和力的磷酸盐, 共聚成 sds-页凝胶17。由于 sds 页凝胶中的 p 标记分子有选择地延缓磷酸化蛋白的电泳迁移, p 标记 sds 页可以将磷酸化蛋白与 non-phosphorylated 的蛋白质分离 (图 1)。如果 protein-of-interest 在多个残留物上磷酸化, 则会观察到相应的磷酸酯形态的阶梯。在 Rab10 的情况下, 我们只观察一个移位带, 表明 Rab10 是磷酸化只在 Thr73。P 标记 SDS 页的主要优势, 免疫与磷酸化特异抗体是, 磷酸化 Rab10 可以检测免疫与 non-phosphorylation 特异抗体 (即, 识别总 Rab10)在被转移的细胞膜之后, 通常是更加具体, 敏感, 并且可利用从商业或学术来源。使用 P 标记 SDS 页的另一个优点是, 可以获得对磷酸化的化学计量的近似估计, 这是不可能的免疫与磷酸化特异性抗体或代谢标记细胞与放射性磷酸盐.
除了使用廉价的 P 标记丙烯酰胺和一些小的修改与它相关, 目前的方法检测 Rab10 磷酸化的 LRRK2 遵循的一般协议免疫。因此, 它应该是直接和易于执行在任何实验室的免疫是一个通常的做法, 与任何类型的样品, 包括纯化蛋白, 细胞裂解, 和组织匀。
Protocol
1. P 标记 SDS–PAGE 的样品制备 从10厘米的盘子中取出并丢弃培养基, 其中细胞是用5毫升 Dulbecco 的磷酸盐缓冲盐水 (DPBS) 的吸入和洗涤细胞生长的, 首先在盘子的一侧加入 DPBS, 以避免干扰细胞层, 然后手工把盘子放回来回几次。 去除和丢弃 DPBS 使用吸入和添加2毫升的 0.25% (瓦特/v) 胰蛋白酶稀释 DPBS, 并轻轻地岩石的盘子覆盖的细胞层。将菜肴放入 co2孵化器 (37 ° c, 加湿空气, 5…
Representative Results
过度表达系统: HA-Rab10 的磷酸化 3×FLAG-LRRK2 在 HEK293 细胞: HEK293 细胞转染0.266 µg HA-Rab10 野生型和1.066 µg 3×FLAG-LRRK2 (野生型, 激酶-不活泼突变体 (K1906M), 或平板变种)。Rab10 磷酸化检查的 P 标记 SDS 页后, 免疫使用抗 HA 抗体 (图 2)。10µg 蛋白质是运行在10% 凝胶 (80 x 100 x 1 毫米) 包含50µM P 标记丙烯酰胺和100µM MnCl<…
Discussion
在这里, 我们描述了一个简便和稳健的方法检测 Rab10 磷酸化的 LRRK2 在内生水平的基础上, P 标记方法。由于目前对磷酸化 Rab10 的抗体仅适用于表达蛋白15, 目前采用 p-标记的方法是评价 Rab10 磷酸化的内源水平的唯一途径。此外, 本方法还允许估计细胞中 Rab10 磷酸化的化学计量。因为 P 标记方法一般适用于磷-蛋白质, 所以本协议可以是为其他磷蛋白的相似方法建立的 “原型”。
<p …Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 Dr. 武 Iwatsubo (日本东京大学) 提供的质粒编码 3 xflag-LRRK2 重量和突变体。我们还感谢 Dr. 达里奥阿莱西 (英国邓迪大学) 提供 MLi-2 和质粒编码 HA-Rab10。这项工作得到了日本促进科学协会 (jsp) KAKENHI 赠款号 JP17K08265 (gi) 的支持。
Materials
| Reagents | |||
| Dulbecco's phosphate-buffered saline (DPBS) | homemade | 150 mM NaCl, 8 mM Na2HPO4-12H2O, 2.7 mM KCl, 1.5 mM KH2PO4 in MilliQ water and sterilized by autoclaving | |
| Sodium chloride | Nacalai Tesque | 31320-34 | |
| Sodium Disodium Hydrogenphosphate 12-Water | Wako | 196-02835 | |
| Potassium chloride | Wako | 163-03545 | |
| Potassium Dihydrogen Phosphate | Wako | 169-04245 | |
| 2.5% Trypsin (10X) | Sigma-Aldrich | T4549 | Dilute 10-fold with sterile DPBS for preparing working solution |
| Dulbecco's modified Eagle medium (DMEM) |
Wako | 044-29765 | |
| Fetal bovine serum | BioWest | S1560 | Heat-inactivated at 56 °C for 30 min |
| Penicillin-Streptomycin (100X) | Wako | 168-23191 | |
| HEPES | Wako | 342-01375 | |
| Sodium hydroxide | Wako | 198-13765 | |
| Polyethylenimine HCl MAX, Linear, Mw 40,000 (PEI MAX 40000) | PolySciences, Inc. | 24765-1 | Stock solution was prepared in 20 mM HEPES-NaOH pH 7.0 at 1 mg/mL and the pH was then adjusted to 7.0 with NaOH |
| Dimethyl sulfoxide | Wako | 045-28335 | |
| Tris | STAR | RSP-THA500G | |
| Hydrochloric acid | Wako | 080-01066 | |
| Polyoxyethylene(10) Octylphenyl Ether | Wako | 160-24751 | Equivalent to Triton X-100 |
| Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) | Wako | 346-01312 | |
| Sodium orthovanadate(V) | Wako | 198-09752 | |
| Sodium fluoride | Kanto Chemical | 37174-20 | |
| β-Glycerophosphoric Acid Disodium Salt Pentahydrate | Nacalai Tesque | 17103-82 | |
| Sodium pyrophosphate decahydrate | Kokusan Chemical | 2113899 | |
| Microcystin-LR | Wako | 136-12241 | |
| Sucrose | Wako | 196-00015 | |
| Complete EDTA-free protease inhibitor cocktail | Roche | 11873580001 | Dissolve one tablet in 1 mL water, which can be stored at -20 °C for a month. Use it at 1:50 dilution for cell lysis |
| Pierce Coomassie (Bradford) Protein Assay Kit | Thermo Fisher Scientific | 23200 | |
| Sodium dodecyl sulfate | Nacalai Tesque | 31607-65 | |
| Glycerol | Wako | 075-00616 | |
| Bromophenol blue | Wako | 021-02911 | |
| β-mercaptoethanol | Kanto Chemical | 25099-00 | |
| Ethanol | Wako | 056-06967 | |
| Methanol | Wako | 136-01837 | |
| Phosphate-binding tag acrylamide | Wako | AAL-107 | P-tag acrylamide |
| 40% (w/v) acrylamide solution | Nacalai Tesque | 06119-45 | Acrylamide:Bis = 29:1 |
| Tetramethylethylenediamine (TEMED) | Nacalai Tesque | 33401-72 | |
| Ammonium persulfate (APS) | Wako | 016-08021 | 10% (w/v) solution was prepared by dissolving the powder of ammonium persulfate in MilliQ water |
| 2-propanol | Wako | 166-04831 | |
| Manganese chloride tetrahydrate | Sigma-Aldrich | M3634 | |
| Precision Plus Protein Prestained Standard | Bio-Rad | 1610374, 1610373, 1610377 | Molecular weight marker used in the protocol |
| WIDE-VIEW Prestained Protein Size Marker III | Wako | 230-02461 | |
| Glycine | Nacalai Tesque | 17109-64 | |
| Amersham Protran NC 0.45 | GE Healthcare | 10600007 | Nitrocellulose membrane |
| Durapore Membrane Filter | EMD Millipore | GVHP00010 | PVDF membrane |
| Filter Papers No.1 | Advantec | 00013600 | |
| Ponceau S | Nacalai Tesque | 28322-72 | |
| Acetic acid | Wako | 017-00251 | |
| Tween-20 | Sigma-Aldrich | P1379 | polyoxyethylenesorbitan monolaurate |
| Ethylenediaminetetraacetic acid (EDTA) | Wako | 345-01865 | |
| Skim milk powder | Difco Laboratories | 232100 | |
| Immunostar | Wako | 291-55203 | ECL solution (Normal sensitivity) |
| Immunostar LD | Wako | 290-69904 | ECL solution (High sensitivity) |
| CBB staining solution | homemade | 1 g CBB R-250, 50% (v/v) methanol, 10% (v/v) acetic acid in 1 L of MilliQ water | |
| CBB R-250 | Wako | 031-17922 | |
| CBB destaining solution | homemade | 12% (v/v) methanol, 7% (v/v) acetic acid in 1 L MilliQ water | |
| Name | Company | Catalog Number | Comments |
| Antibodies | |||
| anti-HA antibody | Sigma-Aldrich | 11583816001 | Used at 0.2 μg/mL for immunoblotting. |
| anti-Rab10 antibody | Cell Signaling Technology | #8127 | Used at 1:1000 for immunoblotting. Specificity was confirmed by CRISPR KO in Ito et al., Biochem J, 2016. |
| anti-pSer935 antibody | Abcam | ab133450 | Used at 1 μg/mL for immunoblotting. |
| anti-LRRK2 antibody | Abcam | ab133518 | Used at 1 μg/mL for immunoblotting. |
| anti-α-tubulin antibody | Sigma-Aldrich | T9026 | Used at 1 μg/mL for immunoblotting. |
| anti-GAPDH antibody | Santa-Cruz | sc-32233 | Used at 0.02 μg/mL for immunoblotting. |
| Peroxidase AffiniPure Sheep Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 515-035-003 | Used at 0.16 μg/mL for immunoblotting. |
| Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 111-035-003 | Used at 0.16 μg/mL for immunoblotting. |
| Name | Company | Catalog Number | Comments |
| Inhibitors | |||
| GSK2578215A | MedChem Express | HY-13237 | Stock solution was prepared in DMSO at 10 mM and stored at -80 °C |
| MLi-2 | Provided by Dr Dario Alessi (University of Dundee) | Stock solution was prepared in DMSO at 10 mM and stored at -80 °C | |
| Name | Company | Catalog Number | Comments |
| Plasmids | |||
| Rab10/pcDNA5 FRT TO HA | Provided by Dr Dario Alessi (University of Dundee) |
This plasmid expresses amino-terminally HA-tagged human Rab10. | |
| LRRK2 WT/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Ito et al., Biochemistry, 46: 1380–1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged wild-type human LRRK2. | |
| LRRK2 K1906M/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Ito et al., Biochemistry, 46: 1380–1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged K1906M kinase-inactive mutant of human LRRK2. | |
| LRRK2 N1437H/p3xFLAG-CMV-10 | This paper. This plasmid expresses amino-terminally 3xFLAG-tagged N1437H FPD mutant of human LRRK2. | ||
| LRRK2 R1441C/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441C FPD mutant of human LRRK2. | |
| LRRK2 R1441G/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441G FPD mutant of human LRRK2. | |
| LRRK2 R1441H/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441H FPD mutant of human LRRK2. | |
| LRRK2 R1441S/p3xFLAG-CMV-10 | This paper. This plasmid expresses amino-terminally 3xFLAG-tagged R1441S FPD mutant of human LRRK2. | ||
| LRRK2 Y1699C/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged Y1699C FPD mutant of human LRRK2. | |
| LRRK2 G2019S/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged G2019S FPD mutant of human LRRK2. | |
| LRRK2 I2020T/p3xFLAG-CMV-10 | Provided by Dr Takeshi Iwatsubo (University of Tokyo) | Kamikawaji et al., Biochemistry, 48: 10963–10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged I2020T FPD mutant of human LRRK2. | |
| Name | Company | Catalog Number | Comments |
| Equipments | |||
| CO2 incubator | Thermo Fisher Scientific | Forma Series II 3110 Water-Jacketed | |
| Auto Pipette | Drummond | Pipet-Aid PA-400 | |
| Micropipette P10 | Nichiryo | 00-NPX2-10 | 0.5–10 μL |
| Micropipette P200 | Nichiryo | 00-NPX2-200 | 20–200 μL |
| Micropipette P1000 | Nichiryo | 00-NPX2-1000 | 100–1000 μL |
| Tips for micropipette P10 | STAR | RST-481LCRST | Sterile |
| Tips for micropipette P200 | FUKAEKASEI | 1201-705YS | Sterile |
| Tips for micropipette P1000 | STAR | RST-4810BRST | Sterile |
| 5 mL disporsable pipette | Greiner | 606180 | Sterile |
| 10 mL disporsable pipette | Greiner | 607180 | Sterile |
| 25 mL disporsable pipette | Falcon | 357535 | Sterile |
| Hematocytometer | Sunlead Glass | A126 | Improved Neubeuer |
| Microscope | Olympus | CKX53 | |
| 10 cm dishes | Falcon | 353003 | For tissue culture |
| 6-well plates | AGC Techno Glass | 3810-006 | For tissue culture |
| Vortex mixer | Scientific Industries | Vortex-Genie 2 | |
| Cell scrapers | Sumitomo Bakelite | MS-93100 | |
| 1.5 mL tubes | STAR | RSV-MTT1.5 | |
| 15 mL tubes | AGC Techno Glass | 2323-015 | |
| 50 mL tubes | AGC Techno Glass | 2343-050 | |
| Centrifuges | TOMY | MX-307 | |
| 96-well plates | Greiner | 655061 | Not for tissue culture |
| Plate reader | Molecular Devices | SpectraMax M2e | |
| SDS–PAGE tanks | Nihon Eido | NA-1010 | |
| Transfer tanks | Nihon Eido | NA-1510B | |
| Gel plates (notched) | Nihon Eido | NA-1000-1 | |
| Gel plates (plain) | Nihon Eido | NA-1000-2 | |
| Silicon spacers | Nihon Eido | NA-1000-16 | |
| 17-well combs | Nihon Eido | Custom made | |
| Binder clips | Nihon Eido | NA-1000-15 | |
| 5 mL syringe | Terumo | SS-05SZ | |
| 21G | Terumo | NN-2138R | |
| Power Station 1000 VC | ATTO | AE-8450 | Power supply for SDS–PAGE and transfer |
| Large weighing boats | Ina Optika | AS-DL | |
| Plastic containers | AS ONE | PS CASE No.4 | 10 x 80 x 50 mm |
| Rocking shaker | Titech | NR-10 | |
| Styrene foam box | generic | The internal dimensions should fit one transfer tank (200 x 250 x 250 mm). | |
| ImageQuant LAS-4000 | GE Healthcare | An imager equipped with a cooled CCD camera for detection of ECL |
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Cite This Article
Ito, G., Tomita, T. Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag. J. Vis. Exp. (130), e56688, doi:10.3791/56688 (2017).