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Oct 2018

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A Radioactive in vitro ERK3 Kinase Assay
体外ERK3激酶的放射性分析   

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Abstract

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that have an important role in signal transduction. Extracellular signal-regulated kinase 3 (ERK3), also known as MAPK6, is an atypical MAPK. Here, we describe in detail an in vitro assay for the kinase activity of ERK3 using myelin basic protein (MBP) or steroid receptor coactivator-3 (SRC-3) as substrates. The assay is carried out in the presence of [γ-32P]-ATP which results in radiolabeling of phosphorylated substrates. Separation of the reaction components by gel electrophoresis followed by autoradiography enables detection of the radiolabeled products, and hence determination of the kinase activity of ERK3. This assay can be used for several applications including identification of substrates, determination of the effect of molecules or mutations on kinase activity, and testing specific kinase inhibitors. Furthermore, the protocol outlined here can be adapted to measure the activity of other kinases by using their specific substrates.

Keywords: ERK3 (ERK3), MAPK (MAPK), Kinase assay (激酶检测), Radioactive assay (放射性分析), 32P-ATP (32P-ATP)

Background

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) (Coulombe and Meloche, 2007). Here we describe an in vitro kinase assay in which ERK3 transfers radiolabeled gamma-phosphate from [γ-32P]-ATP to a purified protein substrate. Excess radiolabeled ATP is then separated from the radiolabeled substrate by gel electrophoresis. The amount of phosphorylated substrate can be quantified by autoradiography, phosphorimaging, or liquid scintillation counting techniques. Radioactive kinase assay provides a direct measurement of kinase activity. It is sensitive, quick, inexpensive, and considered as the 'gold standard' for quantification of protein kinase activity. The major limitations of radioactive kinase assays are the hazards of handling radiolabeled isotopes and unsuitability of this assay format for large scale high-throughput screening.

This protocol describes a direct kinase assay for ERK3 using myelin basic protein (MBP) or a fragment of steroid receptor coactivator-3 (SRC-3) as substrates. MBP is a non-specific substrate for several kinases including members of MAPK family (Haubrich and Swinney, 2016). SRC-3 was shown to interact with ERK3 and is phosphorylated by ERK3 on its Ser857 residue within the CBP-interacting domain (CID) (Long et al., 2012). Hence, SRC3-CID fragment, which comprises amino acids 841-1080, is used as a substrate in this assay.

Another substrate for ERK3 that has been well-characterized and validated to be physiologically relevant is MAPK-activated protein kinase 5 (MK5) (Schumacher et al., 2004, Seternes et al., 2004). ERK3 phosphorylates MK5 at Thr182, leading to MK5 activation. Since MK5 itself is also a kinase, the activity of ERK3 towards MK5 has been determined by a coupled kinase assay in which the phosphorylation of peptide or protein substrate for MK5 is measured in the presence of both ERK3 and MK5 (Schumacher et al., 2004, Seternes et al., 2004).

The in vitro kinase assay described here has been previously used to identify novel ERK3 substrates (Bian et al., 2016), to determine the effect of mutations on the kinase activity of ERK3 (Alsaran et al., 2017, Elkhadragy et al., 2018), and to compare autophosphorylation of wild type or mutant ERK3 (Elkhadragy et al., 2018). In these studies, ERK3 protein was expressed and purified from bacteria, Sf9 insect cells, or mammalian cells. HA-tagged ERK3 expressed and immunoprecipitated from mammalian 293T cells showed greater in vitro kinase activity as compared to recombinant His-tagged ERK3 purified from E. coli (Elkhadragy et al., 2018), possibly because of the greater extent of post-translational modifications or the presence of interacting partners in mammalian cells. Selection of the method for purifying ERK3 protein to be used in a kinase assay has to be based on the purpose and specific considerations of the experiment to be conducted.

Materials and Reagents

  1. Pipette tips
  2. Eppendorf tubes
  3. X-ray film
  4. Filter paper (Bio-Rad Laboratories, catalog number: 1703965)
  5. ERK3 protein: Wild type or mutant ERK3 proteins can be purified from Sf9 insect cells, mammalian cells, or E. coli as described previously (Bian et al., 2016, Elkhadragy et al., 2018)
  6. Recombinant protein substrates: GST-SRC3-CID can be purified as described previously (Elkhadragy et al., 2018), and Myelin Basic Protein (MPB) is commercially available (Millipore Sigma, catalog number: M1891)
  7. [γ-32P]-ATP (Perkin Elmer, catalog number: NEG002Z), stored at -20 °C, Half-life is 14.29 days
  8. ATP (non-radioactive, Thermo Fisher Scientific, catalog number: PV3227)
  9. Phosphatase inhibitor (Sigma-Aldrich, catalog number: P0044)
  10. Magnesium chloride (MgCl2, Thermo Fisher Scientific, catalog number: AM9530G)
  11. Dithiothreitol (DTT, Thermo Fisher Scientific, catalog number: P2325)
  12. Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, Boston BioProducts, catalog number: BM-151)
  13. InstantBlue Coomassie Protein Stain (Expedeon, catalog number: ISB1L), stored at 4 °C
  14. Sodium dodecyl sulfate (SDS, Millipore Sigma, catalog number: 71725)
  15. SDS polyacrylamide gel (Precast or handcast gels can be used)
  16. Tris-HCl (Thermo Fisher Scientific, catalog number: 15567027) 
  17. Beta-mercaptoethanol (Millipore Sigma, catalog number: M3148)
  18. Glycerol (Millipore Sigma, catalog number: G5516)
  19. Bromophenol blue (Millipore Sigma, catalog number: B3269)
  20. 10x kinase reaction buffer (see Recipes), stored at -20 °C in small aliquots
  21. 4x SDS sample buffer (see Recipes), stored at -20 °C in small aliquots

Equipment

  1. Pipettes
  2. -80 °C freezer
  3. Vertical mini-gel electrophoresis system (such as Mini-PROTEAN Tetra Vertical Electrophoresis Cell, Bio-Rad, catalog number: 1658005)
  4. Electrophoresis power supply (such as PowerPac Basic power supply, Bio-Rad, catalog number: 1645050)
  5. Perspex shielding and Perspex Eppendorf tube holders
  6. Geiger counter
  7. Heat blocks or water baths set to 30 °C and 95 °C
  8. Benchtop centrifuge 
  9. Gel dryer (such as Bio-Rad gel dryer Model 583)
  10. X-ray film processor (such as the one from Konica, catalog number: SRX101A) placed in a dark room
  11. X-ray film cassette and security bag
  12. Scanner

Software

  1. ImageJ (National Institutes of Health and the Laboratory for Optical and Computational Instrumentation, USA, imagej.nih.gov/ij)

Procedure

This assay is performed by mixing ERK3 protein, purified substrate, 5 μCi [γ-32P]-ATP, and 30 μM non-radioactive ATP in a kinase reaction buffer that contains Mg2+. Fifty to one hundred nanograms of ERK3 protein purified from Sf9 cells or mammalian cells can be used per reaction. If using ERK3 protein purified from bacteria, a larger amount has to be used (500 ng-1 µg). It is best to do an initial optimization experiment using different amounts of the kinase to determine the kinase concentration appropriate for phosphorylating substrate. A catalytically-inactive (kinase-dead) mutant of ERK3 can serve as a negative control for the reaction. In addition, a reaction that lacks ERK3 would confirm the absence of contamination of the substrate with radioactive isotopes or co-purified kinases. As for all enzymatic assays, the amount of substrate used in each reaction has to be in excess so that it is not rate-limiting. Typically about 0.5-2 µg substrate is used per reaction.
  After incubation of ERK3 with the substrate in the presence of ATP, the reaction is stopped by addition of sample buffer containing sodium dodecyl sulfate (SDS) followed by boiling. Reaction components are separated by gel electrophoresis. The gel is then stained with Coomassie blue solution, dried and visualized by autoradiography (Figure 1).


Figure 1. Outline of radioactive in vitro ERK3 kinase assay

Steps of the procedure are described below:

  1. Prepare a 10x kinase reaction buffer stock (listed in Recipes), and dilute an appropriate volume as needed with deionized water to 1x concentration. 
  2. Dilute ERK3 protein and each purified substrate in 1x reaction buffer such that 1 μl of each is used per reaction. For example, if 100 ng ERK3 protein and 1 μg MBP will be used in the assay, dilute ERK3 to 100 ng/μl, and MBP to 1 μg/μl using 1x reaction buffer.
    Note: It is recommended to store purified proteins at -80 °C in small aliquots to avoid multiple freeze-thaws. Thawing has to be done slowly on ice before an experiment is to be performed.
  3. Prepare 0.5 mM non-radioactive ATP solution by diluting stock ATP in 1x reaction buffer. It is best to prepare fresh dilute ATP at the time of the experiment, and to avoid repeated freeze-thaws of the stock ATP.
  4. Determine the volume of each component to be used such that the total reaction volume is 30 μl (Table 1). The ATP mix added per reaction contains 5 μCi [γ-32P]-ATP and non-radioactive ATP that yields final concentration of 30 μM. For example, ATP mix for one reaction can be prepared by mixing 0.5 μl of radioactive ATP (10 μCi/μl) and 1.8 μl of 0.5 mM non-radioactive ATP.
    Note: The rate of decay of [γ-32P]-ATP has to be taken into consideration when calculating the volume required per reaction. Manufacturers specify a calibration or reference date, which corresponds to the indicated activity of a radiolabeled reagent. This date can be used to determine the residual activity of the radioactive isotope on the day the assay is conducted. For example, for an experiment to be done two weeks after the [γ-32P]-ATP reference date, since the half-life of 32P isotopes is 14.29 days, double the volume of radiolabeled ATP has to be used to account for the decreased activity. Hence, instead of mixing 1.8 μl of 0.5 mM non-radioactive ATP with 0.5 μl of radioactive ATP as suggested above, 1.8 μl of 0.5 mM non-radioactive ATP should be mixed with 1 μl of radioactive ATP. The total volume of each reaction should always be adjusted to 30 µl. 

    Table 1. Components of each kinase reaction


  5. Assemble reactions on ice by mixing all the components except for the ATP mix. If multiple reactions will use the same substrate, a master mix comprising 10x reaction buffer, substrate, and water can be prepared and distributed to the tubes to minimize pipetting errors.
  6. From this step onwards, precautions of using radioactive isotopes have to be taken. Prepare sufficient ATP mix for the number of assays to be performed. Start the reactions by adding an appropriate volume of ATP mix to each tube.
    Note: Radioactive material handling precautions have to be taken. These include the use of appropriate shielding materials such as Perspex shielding (3/8 inches thick) behind which all work should be done, and Perspex Eppendorf tube holders. Surfaces should be routinely monitored by Geiger counters, and ring dosimeters can be used to monitor personal exposure. Radioactive isotopes should be used only by authorized personnel in designated places following the institution’s biosafety regulations. Requisition and storage of radioactive material, solid and liquid radioactive waste disposal, and spill decontamination should be done following the institution’s regulations.
  7. Incubate the tubes in a water bath or sand bath at 30 °C for 30 min.
  8. Stop the reactions by adding 10 μl of 4x SDS sample buffer (listed in Recipes) followed by boiling at 95 °C for 5 min. Spin down the tubes to bring the reaction components to the bottom of the tube.
  9. Load the entire volume of each reaction into a well of a 10-well polyacrylamide gel. The percentage of resolving gel depends on the molecular weight of protein substrates used in the assay. Run the gel at 100-130 constant voltage. It is important to stop gel running when the dye front is about 1-2 cm away from the bottom of the gel to prevent the entry of free [γ-32P]-ATP into the buffer in the gel tank. Cut the gel just above the dye front and discard the lower portion into solid radioactive waste.
  10. Stain the gel with a sufficient volume of InstantBlue Coomassie protein stain for 30 min, followed by two washes using deionized water for 30 min each. Alternatively, standard Coomassie blue staining and destaining solutions can be used. Discard the solutions used for staining and washing into a liquid radioactive waste container.
  11. Place the gel on a thick filter paper, and dry it in a gel dryer at 70 °C for 60 min. 
  12. Expose radioactivity with X-ray film for an appropriate time as follows:
    1. In a dark room place the gel in an X-ray film cassette with an X-ray film directly above it. 
    2. Close the cassette and put it in an X-ray cassette security bag to ensure light protection. The use of intensifying screens enhances the signal. Also, keeping the cassette in a -80 °C freezer enhances the signal. 
    3. Depending on the activity of the kinase towards the specific substrate (the phosphorylation level of the substrate), an X-ray film may be exposed for as short as 2-3 h, or as long as 1-2 days. 
  13. Develop and fix the X-ray film to visualize substrate phosphorylation by autoradiography. Dark bands appearing on the X-ray film correspond to phosphorylated substrate that is isotope-labeled. Several exposures can be obtained to ensure that the signal is not over-saturated.
  14. A representative image of an in vitro ERK3 kinase assay using MBP as substrate is shown in Figure 2. The first lane is a negative control reaction which lacks ERK3. The second lane is a reaction that contains ERK3 and MBP. Note that ERK3 protein is barely seen in the Coomassie-stained gel because of its small amount, whereas the appearance of phosphorylated ERK3 band in the autoradiograph indicates ERK3 autophosphorylation.


    Figure 2. Representative image of in vitro ERK3 kinase assay using MBP as substrate. ERK3 protein with an N-terminal HA-tag (HA-ERK3) was expressed in 293T cells. HA-ERK3 protein was then purified by immunoprecipitation using HA antibody-conjugated beads, followed by elution with HA peptide. In vitro kinase reaction was performed in the presence of [γ-32P]-ATP and MBP (1 µg), with or without 100 ng purified ERK3 (lanes 2 and 1 respectively). Total protein levels of MBP in both reactions are shown by Coomassie staining (left panel). Phosphorylation of MBP is detected by autoradiography (right panel).

Data analysis

Data is analyzed by scanning the dry gel and the developed X-ray film using a standard scanner, and determination of the intensity of bands using image analysis software such as ImageJ. Quantification of substrate phosphorylation is done by calculating the ratio of the band intensity of phosphorylated substrate in the autoradiograph over that of the corresponding total substrate protein in the coomassie-stained gel. The ratio calculated for several samples can each be normalized to a reference condition. A hypothetical example to demonstrate data analysis is shown in Table 2:

Table 2. Demonstration of in vitro kinase assay data analysis

Recipes

  1. 10x kinase reaction buffer (1 ml)
    Components
    Final concentration
    400 μl 1 M Tris-HCl pH 7.5
    400 mM Tris-HCl pH 7.5
    100 μl 1 M MgCl2
    100 mM MgCl2
    10 μl 100 mM EGTA
    1 mM EGTA
    10 μl 1 M DTT
    10 mM DTT
    10 μl phosphatase inhibitor
    1%
    Deionized water
    Add up to 1 ml
  2. 4x SDS sample buffer (10 ml)
    Components
    Final concentration
    2.5 ml 1 M Tris-HCl pH 6.8
    250 mM Tris-HCl pH 6.8
    4 ml 100% glycerol
    40% glycerol
    0.8 g SDS
    8% SDS
    40 mg bromophenol blue
    0.4% bromophenol blue
    0.5 ml beta-mercaptoethanol
    5% beta-mercaptoethanol
    Deionized water
    Add up to 10 ml

Acknowledgments

This protocol was modified from the research article by Cheng et al. (1996). This work was supported by a start-up fund of Wright State University and NCI 1R01CA193264-01 to Weiwen Long, and by the Biomedical Sciences PhD Program of Wright State University to Lobna Elkhadragy.

Competing interests

The authors declare no conflict of interest.

References

  1. Alsaran, H., Elkhadragy, L., Shakya, A. and Long, W. (2017). L290P/V mutations increase ERK3's cytoplasmic localization and migration/invasion-promoting capability in cancer cells. Sci Rep 7(1): 14979.
  2. Bian, K., Muppani, N. R., Elkhadragy, L., Wang, W., Zhang, C., Chen, T., Jung, S., Seternes, O. M. and Long, W. (2016). ERK3 regulates TDP2-mediated DNA damage response and chemoresistance in lung cancer cells. Oncotarget 7(6): 6665-6675.
  3. Cheng, M., Boulton, T. G. and Cobb, M. H. (1996). ERK3 is a constitutively nuclear protein kinase. J Biol Chem 271(15): 8951-8958.
  4. Coulombe, P. and Meloche, S. (2007). Atypical mitogen-activated protein kinases: structure, regulation and functions. Biochim Biophys Acta 1773(8): 1376-1387.
  5. Elkhadragy, L., Alsaran, H., Morel, M. and Long, W. (2018). Activation loop phosphorylation of ERK3 is important for its kinase activity and ability to promote lung cancer cell invasiveness. J Biol Chem 293(42): 16193-16205.
  6. Haubrich, B. A. and Swinney, D. C. (2016). Enzyme activity assays for protein kinases: strategies to identify active substrates. Curr Drug Discov Technol 13(1): 2-15.
  7. Long, W., Foulds, C. E., Qin, J., Liu, J., Ding, C., Lonard, D. M., Solis, L. M., Wistuba, II, Qin, J., Tsai, S. Y., Tsai, M. J. and O'Malley, B. W. (2012). ERK3 signals through SRC-3 coactivator to promote human lung cancer cell invasion. J Clin Invest 122(5): 1869-1880.
  8. Schumacher, S., Laass, K., Kant, S., Shi, Y., Visel, A., Gruber, A. D., Kotlyarov, A. and Gaestel, M. (2004). Scaffolding by ERK3 regulates MK5 in development. EMBO J 23(24): 4770-4779.
  9. Seternes, O. M., Mikalsen, T., Johansen, B., Michaelsen, E., Armstrong, C. G., Morrice, N. A., Turgeon, B., Meloche, S., Moens, U. and Keyse, S. M. (2004). Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway. EMBO J 23(24): 4780-4791.

简介

丝裂原活化蛋白激酶(MAPK)是丝氨酸/苏氨酸激酶,其在信号转导中具有重要作用。细胞外信号调节激酶3(ERK3),也称为MAPK6,是非典型MAPK。在这里,我们详细描述了使用髓鞘碱性蛋白(MBP)或类固醇受体辅激活因子-3(SRC-3)作为底物的ERK3激酶活性的体外测定。该测定在[γ- 32 P] -ATP存在下进行,这导致磷酸化底物的放射性标记。通过凝胶电泳然后放射自显影分离反应组分使得能够检测放射性标记的产物,并因此确定ERK3的激酶活性。该测定可用于多种应用,包括底物的鉴定,分子或突变对激酶活性的影响的测定,以及测试特异性激酶抑制剂。此外,此处概述的方案可以适用于通过使用其特定底物来测量其他激酶的活性。
【背景】细胞外信号调节激酶3(ERK3)是非典型促分裂原活化蛋白激酶(MAPK)(Coulombe和Meloche,2007)。在这里,我们描述了体外激酶测定,其中ERK3将放射性标记的γ-磷酸从[γ- 32 P] -ATP转移至纯化的蛋白质底物。然后通过凝胶电泳将过量的放射性标记的ATP与放射性标记的底物分离。磷酸化底物的量可通过放射自显影,磷光成像或液体闪烁计数技术来量化。放射性激酶测定提供激酶活性的直接测量。它灵敏,快速,廉价,被认为是量化蛋白激酶活性的“黄金标准”。放射性激酶测定的主要限制是处理放射性标记的同位素的危险和该测定形式的不适合用于大规模高通量筛选。
该方案描述了使用髓鞘碱性蛋白(MBP)或类固醇受体辅激活因子-3(SRC-3)的片段作为底物的ERK3的直接激酶测定。 MBP是几种激酶的非特异性底物,包括MAPK家族的成员(Haubrich和Swinney,2016)。 SRC-3显示与ERK3相互作用,并在CBP相互作用结构域(CID)内的Ser 857 残基上被ERK3磷酸化(Long et al。,2012) 。因此,在该测定中使用包含氨基酸841-1080的SRC3-CID片段作为底物。
ERK3的另一种底物已被充分表征并经过验证,具有生理学相关性,是MAPK活化蛋白激酶5(MK5)(Schumacher et al。,2004,Seternes et al。,2004)。 ERK3在Thr 182 处磷酸化MK5,导致MK5活化。由于MK5本身也是一种激酶,ERK3对MK5的活性已经通过偶联激酶试验确定,其中在ERK3和MK5存在下测量MK5的肽或蛋白质底物的磷酸化(Schumacher 等人。,2004,Seternes et al。,2004)。
此处描述的体外激酶测定法先前已用于鉴定新型ERK3底物(Bian et al。,2016),以确定突变对激酶活性的影响。 ERK3(Alsaran et al。,2017,Elkhadragy et al。,2018),并比较野生型或突变型ERK3的自身磷酸化(Elkhadragy et al。< / em>,2018)。在这些研究中,从细菌,Sf9昆虫细胞或哺乳动物细胞表达并纯化ERK3蛋白。与从大肠杆菌中纯化的重组His标记的ERK3相比,从哺乳动物293T细胞表达并免疫沉淀的HA标记的ERK3显示出更大的体外激酶活性(Elkhadragy et al。, 2018),可能是因为更大程度的翻译后修饰或哺乳动物细胞中相互作用配偶体的存在。纯化用于激酶测定的ERK3蛋白的方法的选择必须基于要进行的实验的目的和具体考虑。

关键字:ERK3, MAPK, 激酶检测, 放射性分析, 32P-ATP

材料和试剂

  1. 移液器吸头
  2. Eppendorf管
  3. X光片
  4. 滤纸(Bio-Rad Laboratories,目录号:1703965)
  5. ERK3蛋白:野生型或突变型ERK3蛋白可以从Sf9昆虫细胞,哺乳动物细胞或 E中纯化。大肠杆菌如前所述(Bian et al。,2016,Elkhadragy et al。,2018)
  6. 重组蛋白质底物:GST-SRC3-CID可如前所述进行纯化(Elkhadragy et al。,2018),髓磷脂碱性蛋白(MPB)可商购(Millipore Sigma,目录号:M1891)
  7. [γ- 32 P] -ATP(Perkin Elmer,目录号:NEG002Z),储存于-20°C,半衰期为14.29天
  8. ATP(非放射性,Thermo Fisher Scientific,目录号:PV3227)
  9. 磷酸酶抑制剂(Sigma-Aldrich,目录号:P0044)
  10. 氯化镁(MgCl 2 ,Thermo Fisher Scientific,目录号:AM9530G)
  11. 二硫苏糖醇(DTT,Thermo Fisher Scientific,目录号:P2325)
  12. 乙二醇 - 双(β-氨基乙基醚)-N,N,N',N'-四乙酸(EGTA,Boston BioProducts,目录号:BM-151)
  13. InstantBlue Coomassie蛋白质染色剂(Expedeon,目录号:ISB1L),储存于4°C
  14. 十二烷基硫酸钠(SDS,Millipore Sigma,目录号:71725)
  15. SDS聚丙烯酰胺凝胶(可以使用预制或手工凝胶)
  16. Tris-HCl(赛默飞世尔科技,目录号:15567027)&nbsp;
  17. β-巯基乙醇(Millipore Sigma,目录号:M3148)
  18. 甘油(Millipore Sigma,目录号:G5516)
  19. Bromophenol blue(Millipore Sigma,目录号:B3269)
  20. 10x激酶反应缓冲液(参见配方),以小等分试样储存于-20°C
  21. 4x SDS样品缓冲液(参见配方),以小等分试样储存于-20°C

设备

  1. 移液器
  2. -80°C冰箱
  3. 立式微型凝胶电泳系统(如Mini-PROTEAN Tetra Vertical Electrophoresis Cell,Bio-Rad,目录号:1658005)
  4. 电泳电源(如PowerPac Basic电源,Bio-Rad,目录号:1645050)
  5. 有机玻璃屏蔽和Perspex Eppendorf管支架
  6. 盖革计数器
  7. 加热块或水浴设置为30°C和95°C
  8. 台式离心机&nbsp;
  9. 凝胶干燥器(如Bio-Rad凝胶干燥器型号583)
  10. X光胶片处理器(如柯尼卡的一个,目录号:SRX101A)放在黑暗的房间里
  11. X光胶片盒和安全袋
  12. 扫描仪

软件

  1. ImageJ(美国国立卫生研究院和光学与计算仪器实验室,imagej.nih.gov/ij)

程序

通过在含有Mg 2+的激酶反应缓冲液中混合ERK3蛋白,纯化的底物,5μCi[γ- 32 P] -ATP和30μM非放射性ATP来进行该测定。每次反应可以使用从Sf9细胞或哺乳动物细胞纯化的50至100纳克ERK3蛋白。如果使用从细菌中纯化的ERK3蛋白,则必须使用更大量(500 ng-1μg)。最好使用不同量的激酶进行初始优化实验,以确定适合磷酸化底物的激酶浓度。 ERK3的催化失活(激酶死亡)突变体可以作为反应的阴性对照。此外,缺乏ERK3的反应将证实没有放射性同位素或共纯化激酶污染底物。对于所有酶测定,每个反应中使用的底物量必须过量,以使其不受速率限制。通常每次反应使用约0.5-2μg底物。
&NBSP;在ATP存在下将ERK3与底物温育后,通过加入含有十二烷基硫酸钠(SDS)的样品缓冲液终止反应,然后煮沸。通过凝胶电泳分离反应组分。然后用考马斯蓝溶液染色凝胶,干燥并通过放射自显影观察(图1)。


图1.放射性体外 ERK3激酶测定的概述

该程序的步骤如下所述:

  1. 准备10x激酶反应缓冲液(在食谱中列出),并根据需要用去离子水稀释适当的体积至1x浓度。&nbsp;
  2. 在1x反应缓冲液中稀释ERK3蛋白和每种纯化的底物,使得每次反应使用1μl。例如,如果在测定中使用100ng ERK3蛋白和1μgMBP,则使用1x反应缓冲液将ERK3稀释至100ng /μl,并将MBP稀释至1μg/μl。
    注意:建议将纯化的蛋白质在-80°C下以小等分试样储存,以避免多次冻融。在进行实验之前,必须在冰上慢慢解冻。
  3. 通过在1x反应缓冲液中稀释原液ATP制备0.5mM非放射性ATP溶液。最好在实验时制备新鲜的稀释ATP,并避免反复冻融原浆ATP。
  4. 确定每种组分的体积,使总反应体积为30μl(表1)。每次反应添加的ATP混合物含有5μCi[γ- 32 P] -ATP和非放射性ATP,最终浓度为30μM。例如,可以通过混合0.5μl放射性ATP(10μCi/μl)和1.8μl0.5mM非放射性ATP来制备用于一个反应的ATP混合物。
    注意:在计算所需体积时,必须考虑[γ - 32 P] -ATP的衰变速率每个反应。制造商指定校准或参考日期,其对应于放射性标记试剂的指示活性。该日期可用于确定进行测定当天放射性同位素的残留活性。例如,在[γ - 32 P] -ATP参考日期后两周进行实验,因为半衰期 32 P同位素的时间为14.29天,必须使用放射性标记的ATP体积的两倍来解释活性降低。因此,如上所述,不要将1.8μl的0.5 mM非放射性ATP与0.5μl放射性ATP混合,而应将1.8μl的0.5 mM非放射性ATP与1μl放射性ATP混合。每次反应的总体积应始终调整为30μl。

    表1.每种激酶反应的成分


  5. 通过混合除ATP混合物之外的所有组分在冰上组装反应。如果多个反应将使用相同的底物,则可以制备包含10x反应缓冲液,底物和水的主混合物并将其分配到管中以最小化移液误差。
  6. 从这一步开始,必须采取放射性同位素的预防措施。准备足够的ATP混合物用于进行的测定数量。通过在每个试管中加入适量的ATP混合物来开始反应。
    注意:必须采取放射性物质处理注意事项。其中包括使用适当的屏蔽材料,如有机玻璃屏蔽(3/8英寸厚),后面应完成所有工作,以及Perspex Eppendorf管架。 Geiger计数器应常规监测表面,并可使用环剂量计监测个人暴露情况。放射性同位素只能由机构生物安全法规规定的指定地点的授权人员使用。应按照机构的规定,对放射性物质,固体和液体放射性废物处理以及溢出物去污进行征用和储存。
  7. 将试管在30℃的水浴或沙浴中孵育30分钟。
  8. 通过加入10μl4xSDS样品缓冲液(在配方中列出)然后在95℃下煮沸5分钟来终止反应。旋转管子使反应组分进入管子底部。
  9. 将每个反应的全部体积加载到10孔聚丙烯酰胺凝胶的孔中。解析凝胶的百分比取决于测定中使用的蛋白质底物的分子量。以100-130恒定电压运行凝胶。当染料前端离凝胶底部约1-2厘米时,重要的是停止凝胶运行,以防止游离的[γ- 32 P] -ATP进入缓冲液中。凝胶罐。在染料正面上方切割凝胶,并将下部丢弃成固体放射性废物。
  10. 用足量的InstantBlue Coomassie蛋白质染色剂将凝胶染色30分钟,然后用去离子水洗涤两次,每次30分钟。或者,可以使用标准的考马斯蓝染色和脱色溶液。将用于染色和洗涤的溶液丢弃到液体放射性废物容器中。
  11. 将凝胶放在厚滤纸上,在70℃的凝胶干燥器中干燥60分钟。&nbsp;
  12. 用X射线胶片暴露放射性适当的时间如下:
    1. 在黑暗的房间里,将凝胶放在X射线胶片暗盒中,正上方有一个X光胶片。&nbsp;
    2. 关闭录像带并将其放入X光盒安全袋中以确保光线保护。增强屏幕的使用增强了信号。此外,将盒式磁带保持在-80°C冰箱中可以增强信号。&nbsp;
    3. 取决于激酶对特定底物的活性(底物的磷酸化水平),X射线胶片可暴露短至2-3小时,或长达1-2天。&nbsp;
  13. 开发并固定X射线胶片,通过放射自显影观察底物磷酸化。出现在X射线胶片上的暗带对应于同位素标记的磷酸化底物。可以获得几次曝光以确保信号不会过饱和。
  14. 使用MBP作为底物的体外 ERK3激酶测定的代表性图像显示在图2中。第一泳道是缺乏ERK3的阴性对照反应。第二泳道是含有ERK3和MBP的反应。注意,在考马斯染色的凝胶中几乎看不到ERK3蛋白,因为其量很小,而放射自显影照片中磷酸化的ERK3条带的出现表明ERK3自身磷酸化。


    图2.使用MBP作为底物的体外 ERK3激酶测定的代表性图像。具有N末端HA标签(HA-ERK3)的ERK3蛋白在293T细胞中表达。然后使用HA抗体缀合的珠子通过免疫沉淀纯化HA-ERK3蛋白质,然后用HA肽洗脱。 体外激酶反应在[γ- 32 P] -ATP和MBP(1μg)存在下进行,有或没有100 ng纯化的ERK3(泳道2和分别为1)。通过考马斯染色显示两个反应中MBP的总蛋白水平(左图)。通过放射自显影检测MBP的磷酸化(右图)。

数据分析

通过使用标准扫描仪扫描干凝胶和显影的X射线胶片来分析数据,并使用诸如ImageJ的图像分析软件确定条带的强度。通过计算放射自显影照片中磷酸化底物的条带强度与考马斯染色凝胶中相应的总底物蛋白质的比率来完成底物磷酸化的定量。对于几个样品计算的比率可以各自标准化为参考条件。表2中显示了一个证明数据分析的假设示例:

表2.体外激酶测定数据分析的证明

食谱

  1. 10x激酶反应缓冲液(1 ml) class =“ke-zeroborder”bordercolor =“#000000”style =“width:450px;” border =“0”cellspacing =“0”cellpadding =“2”>组件
    最终集中
    400μl1MTris-HCl pH 7.5
    400mM Tris-HCl pH 7.5
    100μl1MMgCl 2
    100mM MgCl 2
    10μl100mM EGTA
    1 mM EGTA
    10μl1M DTT
    10 mM DTT
    10μl磷酸酶抑制剂
    1%
    去离子水
    加入1毫升
  2. 4x SDS样品缓冲液(10 ml) class =“ke-zeroborder”bordercolor =“#000000”style =“width:450px;” border =“0”cellspacing =“0”cellpadding =“2”>组件
    最终集中
    2.5ml 1M Mris-HCl pH 6.8
    250mM Tris-HCl pH 6.8
    4毫升100%甘油
    40%甘油
    0.8克SDS
    8%SDS
    40毫克溴酚蓝
    0.4%溴酚蓝
    0.5毫升β-巯基乙醇
    5%β-巯基乙醇
    去离子水
    加入10毫升

致谢

该协议是由Cheng et al。(1996)的研究文章修改而来的。这项工作得到了赖特州立大学的启动基金和NCI 1R01CA193264-01给魏文龙的支持,以及莱特州立大学生物医学科学博士项目给Lobna Elkhadragy的支持。

利益争夺

作者宣称没有利益冲突。

参考

  1. Alsaran,H.,Elkhadragy,L.,Shakya,A。和Long,W。(2017)。 L290P / V突变增加了ERK3在癌细胞中的细胞质定位和迁移/侵袭促进能力。 Sci Rep 7(1):14979。
  2. Bian,K.,Muppani,N.R.,Elkhadragy,L.,Wang,W.,Zhang,C.,Chen,T.,Jung,S.,Seternes,O。M. and Long,W。(2016)。 ERK3调节肺癌细胞中TDP2介导的DNA损伤反应和化疗耐药性。 Oncotarget 7(6):6665-6675。
  3. Cheng,M.,Boulton,T。G.和Cobb,M。H.(1996)。 ERK3是一种组成型核蛋白激酶。 J Biol Chem 271(15):8951-8958。
  4. Coulombe,P。和Meloche,S。(2007)。 非典型丝裂原活化蛋白激酶:结构,调节和功能。 Biochim Biophys Acta 1773(8):1376-1387。
  5. Elkhadragy,L.,Alsaran,H.,Morel,M。和Long,W。(2018)。 激活环磷酸化ERK3对其激酶活性和促进肺癌细胞侵袭能力非常重要。< / a> J Biol Chem 293(42):16193-16205。
  6. Haubrich,B。A.和Swinney,D。C.(2016)。 蛋白激酶的酶活性测定:识别活性底物的策略。 Curr药物Discov Technol 13(1):2-15。
  7. Long,W.,Foulds,CE,Qin,J.,Liu,J.,Ding,C.,Lonard,DM,Solis,LM,Wistuba,II,Qin,J.,Tsai,SY,Tsai,MJ和O 'Malley,BW(2012)。 ERK3通过SRC-3辅激活因子发出信号,促进人类肺癌细胞的侵袭。 J Clin Invest 122(5):1869-1880。
  8. Schumacher,S.,Laass,K.,Kant,S.,Shi,Y.,Visel,A.,Gruber,A.D.,Kotlyarov,A。和Gaestel,M。(2004)。 ERK3的脚手架对MK5进行了开发。 EMBO J 23(24):4770-4779。
  9. Seternes,O.M.,Mikalsen,T.,Johansen,B.,Michaelsen,E.,Armstrong,C.G.,Morrice,N.A.,Turgeon,B.,Meloche,S.,Moens,U。和Keyse,S.M。(2004)。 非典型MAP激酶ERK3对MK5 / PRAK的激活定义了一种新的信号转导途径。 EMBO J 23(24):4780-4791。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Elkhadragy, L. and Long, W. (2019). A Radioactive in vitro ERK3 Kinase Assay. Bio-protocol 9(16): e3332. DOI: 10.21769/BioProtoc.3332.
  2. Elkhadragy, L., Alsaran, H., Morel, M. and Long, W. (2018). Activation loop phosphorylation of ERK3 is important for its kinase activity and ability to promote lung cancer cell invasiveness. J Biol Chem 293(42): 16193-16205.
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