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In vitro mTORC1 Kinase Assay for Mammalian Cells Protocol
哺乳动物细胞的体外mTORC1激酶酶活测试法   

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Abstract

Historically, mechanistic target of rapamycin (mTOR) was purified from mammalian cells using mild nonionic detergents such as NP-40 and or Triton-X100 that resulted in dissociation of core regulatory components essential for its native kinase activity. Consequently, these older kinase assays required MnCl2 to artificially enhance the weak phosphotransfer activity observed (Bai et al., 2007; Kim et al., 2002). With the use of the zwitterionic detergent 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), the mTOR complex 1 (mTORC1) containing Regulatory-associated protein of mTOR (Raptor) and Lst8 (also known as GbetaL) can be successfully purified as a complex. This in vitro kinase assay allows for purification of mTORC1 that resembles its physiological state and retains kinase activity under physiological MgCl2 concentrations (Sancak et al., 2007). The activity of mTORC1 can be further enhanced through the use of hyperactive mutations within the kinase domain of mTOR or inclusion of GTP-bound RAS enriched in brain (Rheb) that is supplemented into the in vitro kinase assays. Rheb is a small-G-protein that binds to and activates mTORC1 to phosphorylate downstream substrates, such as eukaryotic initiation factor 4E-BP1 (4E-BP1) (Burnett et al., 1998), ribosomal protein S6 kinase 1 (S6K1) (Kim et al., 2002), Signal transducer and activator of transcription 3 (STAT3) (Dodd et al., 2015), and proline-rich Akt substrate of 40 kDa (PRAS40) (Dunlop et al., 2009).

Materials and Reagents

  1. HEK293E cells
  2. Plasmids and vectors
    1. HA-Raptor (Addgene, catalog number: 8513 )
    2. myc-mTOR (Addgene, catalog number: 1861 )
    3. Rheb (National Center for Biotechnology Information, Gene, catalog number: 6009 ) cloned into pDEST27 using the gateway cloning system in accordance with manufacturer protocol (Life Technologies, catalog number: 11812-013 )
      Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: 11812-013 ”.
    4. GST-4E-BP1/pGEX vectors generated as previously described (Dunlop et al., 2009)
  3. Antibodies
    1. Clone 9E10 anti-Myc antibodies (Sigma-Aldrich, catalog number: M5546 )
    2. Clone 9B11 anti-Myc antibodies (Cell Signaling Technology, catalog number: 2276 )
    3. Anti-HA (Roche Diagnostics, catalog number: 11867431001 )
    4. Anti-GST (Merck Millipore Corporation, catalog number: 05-782 )
  4. Cell culture and transfection
    1. Dulbecco’s modified eagle’s medium (DMEM)
    2. 10% foetal bovine serum (FBS), EU Approved (South American) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
    3. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15070-063 )
      Note: HEK293E cells were cultured in DMEM supplemented with 10% FBS, 1 μg/ml penicillin and 1 µg/ml streptomycin.
  5. Insulin (Sigma-Aldrich, catalog number: I9278 )
  6. Rapamycin (EMD Millipore Corporation, catalog number: 553210 )
  7. Chemicals of analytical grade
    1. HEPES (Sigma-Aldrich, catalog number: H3375 )
    2. EDTA (Sigma-Aldrich, catalog number: 431788 )
    3. β-glycerophosphate (disodium salt, pentahydrate) (Sigma-Aldrich, catalog number: 50020 )
    4. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
    5. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
    6. Adenine triphosphate (ATP) (Sigma-Aldrich, catalog number: A26209 )
    7. Leupeptin (Sigma-Aldrich, catalog number: L5793 )
    8. Antipain (Sigma-Aldrich, catalog number: 10791 )
    9. Benzamadine (Sigma-Aldrich, catalog number: 12072 )
    10. Pepstatin A (Sigma-Aldrich, catalog number: P5318 )
    11. Sodium vanadate (Sigma-Aldrich, catalog number: 289361 )
    12. Dithiothreitol (Sigma-Aldrich, catalog number: 43815 )
    13. Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: 78830 )
    14. Wortmannin (Sigma-Aldrich, catalog number: W1628 )
    15. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) (Sigma-Aldrich, catalog number: 226947 )
  8. mTOR lysis buffer (see Recipes)
  9. Low salt mTOR wash buffer (see Recipes)
  10. High salt mTOR wash buffer (see Recipes)
  11. mTOR wash buffer (see Recipes)
  12. 3x mTOR kinase assay buffer (see Recipes)
  13. Rheb lysis buffer (see Recipes)
  14. Rheb storage buffer (see Recipes)
  15. Phosphate buffered saline (see Recipes)
  16. mTOR assay start buffer (see Recipes)
  17. Protease inhibitors (see Recipes)

Equipment

  1. Thermo mixer heating block (Eppendorf AG, model: Eppendorf thermomixer® compact )
  2. Refrigerated mini centrifuge (Thermo Fisher Scientific, Thermo Scientific™, model: Heraeus and Fresco 17 centrifuge )
  3. StuartTM SB2 fixed speed rotator (Bibby Scientific Limited, Stuart Scientific)

Procedure

Note: An overview of the whole procedure can be found in Figure 1.



Figure 1. mTOR in vitro kinase assay. As described in the “Procedure”, preparation of (A) mTORC1 complexes, (B) Rheb-GTP and (D) drug inhibtors (rapamycin/FKBP12 and wortmannin) and incubation of mTORC1 substrate (4E-BP1) within the mTORC1 kinase assay (E).

  1. Generating mTOR/raptor complexes from HEK293E cells
    1. HEK293E cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1%, 100 μg/ml penicillin and 100 µg/ml streptomycin.
    2. 75 cm2 flasks of 80% confluent HEK293E cells were either co-transfected with 5 µg Myc-tagged mTOR and 5 µg HA-tagged Raptor constructs or transfected with a GST-tagged Rheb construct using the calcium phosphate transfection method (1 x 75 cm2 flask of HEK293E cells is sufficient for three mTOR kinase assays). Cells were harvested 36 h post-transfection. Cells are treated with 10 µg/ml insulin for 30 min prior to lysis. This dose is sufficient to stimulate mTORC1 signaling and ensure that the active complex is purified as previously demonstrated (Dunlop et al., 2009).
    3. Stimulate cells with 100 nM insulin for 15 min then lyse in 1 ml of mTOR lysis buffer supplemented with protease inhibitors and 0.3% CHAPS (w/v).
    4. Centrifuge at 16,200 x g for 8 min at 4 °C (refrigerated mini centrifuge).
    5. Incubate lysates with 3 μl of Myc- or HA-antibodies (the mTORC1 complex can be purified with either HA-raptor or myc-mTOR immunoprecipitation) for 1.5 h at 4 °C with rotation.
    6. Make a 50% volume ratio mix of Protein G plus mTOR lysis buffer, add 40 μl to each tube and incubate for 1 h at 4 °C with rotation (StuartTM SB2 fixed speed rotator).
    7. Wash immunoprecipitates with 0.5 ml of the following buffers supplemented with protease inhibitors:
      1. 1x low salt mTOR wash buffer (supplemented with 0.3% w/v CHAPS)
      2. 2x high salt mTOR wash buffer (supplemented with 0.3% w/v CHAPS)
      3. 2x mTOR wash buffer
    8. Split immunoprecipitates equally into three Eppendorf tubes for the in vitro kinase assay.

  2. Preparing GTP-bound Rheb
    Human Rheb (Gene ID: 6009) cloned into pDEST27, which contains an N-terminal GST-tag.
    1. Transfect four 75 cm2 flasks of 80% confluent HEK293E cells with GST-Rheb-pDEST27 (10 µg DNA per flask), using standard calcium phosphate transfection procedures (Schalm et al., 2002).
    2. Grow HEK293E cells over-night in the presence of 10% (v/v) FBS till fully confluent (6-7 x 106 cells).
    3. Lyse HEK293E cells in 1 ml Rheb lysis buffer supplemented with 0.3% (w/v) CHAPS (plus protease inhibitors).
      Note: Reducing agents (such as DTT) should be omitted as this will interfere with GST-purification. Incubate lysates on ice for 30 min to facilitate lysis.
    4. Incubate pre-cleared lysates (after centrifugation at 16,200 x g for 10 min at 4 °C) with immobilized 40 μl of a 50% volume ratio mix of Rheb lysis buffer and glutathione-sepharose beads, incubate for 2 h at 4 °C with rotation.
    5. Wash the glutathione-sepharose beads twice with 0.5 ml Rheb lysis buffer and then once with 0.5 ml Rheb storage buffer supplemented with protease inhibitors.
    6. Elute GST-Rheb from the glutathione-sepharose beads in 50 µl Rheb storage buffer supplemented with of 10 mM glutathione (pH readjusted back to 8.0).
    7. Incubate eluted GST-Rheb protein at 30 °C for 10 min with either 10 mM EDTA and 1 mM GDP to generate inactive Rheb-GDP, or 10 mM EDTA and 0.1 mM non-hydrolysable GTPγS to generate active Rheb-GTP. To tightly bind the guanine nucleotide to Rheb, add MgCl2 to a final concentration of 20 mM. Incubate on ice until use.

  3. Generating dephosphorylated 4E-BP1 protein for positive control substrate
    1. Transform BL21 (DE3) pLys bacteria with GST-tagged 4E-BP1/pGEX plasmid (4E-BP1 GeneID, 1978) using standard transformation methods.
    2. Grow bacteria until OD600 is 0.6-0.8, to induce expression add isopropyl-β D-thiogalactoside (IPTG) to give a final concentration of 0.5 mM and incubate for 3 h at 30 °C. Pellet cells by centrifugation at 1,500 x g for 30 min at 4 °C.
    3. Lyse bacteria with a freeze/thaw cycle in 10 ml of PBS supplemented with 10 mM EDTA, 0.1% (v/v) Triton and protease inhibitors.
    4. Use pulse sonication to shear bacterial DNA [3 x 5 sec cycles on full power (30 μm)]. Centrifuge at 16,200 x g for 10 min at 4 °C, then purify GST-4E-BP1 from the bacterial supernatant using glutathione-sepharose beads.
    5. Dephosphorylate GST-4E-BP1 protein using 50 U shrimp alkaline phosphatase, washed in PBS, 10 mM EDTA, 0.1% (v/v) Triton X-100. Elute in 10 mM reduced glutathione in PBS (pH 7.6).
    6. Desalt the eluent using a HiTrap Desalting Column in accordance with manufacturer protocol.
    7. Resolve using SDS-PAGE and stain with Coomassie Brilliant Blue to check the purity and concentration against known bovine serum albumin (BSA) standards.
    8. Dephosphorylated 4E-BP1 can be stored at -80 °C in 10 µg/µl aliquots for future use.

  4. Preparing FKBP12/rapamycin drug/protein complexes to inhibit mTORC1
    1. Human FKBP12 protein can be expressed and purified using the same protocol to generate GST-4E-BP1 above (omitting the dephosphorylation step C5).
    2. FKBP12 can also be frozen in 10 µg/µl aliquots at -80 °C for future use.
    3. To generate FKBP12/rapamycin complexes: Make up 25 mM HEPES (pH 7.4), 10 mM MgCl2, 20 mM rapamycin and 0.5 µg FKBP12 in 10 µl final volume (dH2O). Incubate at room temperature in the dark for 5 min, and store on ice until needed.

  5. Performing mTOR kinase assays
    1. Make up mTOR/Raptor immunoprecipitates in 3x mTOR kinase assay buffer and add 75 ng of Rheb-GTP and or 2 μl FKBP12/rapamycin as required.
    2. Incubate for 20 min on ice prior to starting the kinase assay.
    3. Add 10 μl of mTOR assay start buffer supplemented with 500 μM ATP (freshly added before use) plus 150 ng of purified GST-4E-BP1 or the test substrate to start the assay. Phospho-4E-BP1 (Thr 36/45) levels are used as a positive control and indicate mTOR kinase activity.
    4. Incubate at 30 °C for 30-60 min in thermo mixer heating block, shaking at20 FCS.
    5. Stop reaction by adding 4x sample buffer.
    6. Analyse samples using SDS-PAGE and western blotting. An example of representative data is shown in Figure 2.

Representative data



Figure 2. mTORC1 directed phosphorylation of 4E-BP1. Western blotting showing phosphorylation of purified GST-4E-BP1 after in vitro mTORC1 kinase assay performed in the presence and absence of GTP-Rheb. Levels of myc-mTOR and HA-Raptor are shown as controls.

Notes

  1. It is essential that Rheb is purified from mammalian cells, rather than from bacteria, as proper folding and post-translational modifications (such as prenylation) is required for its activity to enhance mTORC1.

Recipes

Note: All buffers are stored at 4 °C unless otherwise stated.

  1. mTOR lysis buffer
    40 mM HEPES (pH 7.4)
    2 mM EDTA
    10 mM β-glycerophosphate
  2. Low salt mTOR wash buffer
    40 mM HEPES (pH 7.4)
    150 mM NaCl
    2 mM EDTA
    10 mM β-glycerophosphate
  3. High salt mTOR wash buffer
    40 mM HEPES (pH 7.4)
    400 mM NaCl
    2 mM EDTA
    10 mM β-glycerophosphate
  4. mTOR wash buffer
    25 mM HEPES (pH 7.4)
    20 mM KCl
  5. 3x mTOR kinase assay buffer (aliquoted and stored at -20 °C)
    X3 stock solution: 75 mM HEPES (pH 7.4), 60 mM KCl, 30 mM MgCl2
    Add 1:50 dilution of 0.5 M stock of MgCl2 to 25 mM HEPES (pH 7.4), 20 mM KCl
  6. Rheb lysis buffer
    40 mM HEPES (pH 7.4)
    10 mM glycerophosphate
    5 mM MgCl2
  7. Rheb storage buffer
    20 mM HEPES (pH 8.0)
    200 mM NaCl
    5 mM MgCl2
  8. Phosphate buffered saline
    137 mM NaCl
    10 mM phosphate
    2.7 mM KCl (pH 7.4)
  9. mTOR assay start buffer (aliquoted and stored at -20 °C)
    25 mM HEPES (pH 7.4)
    10 mM MgCl2
    140 mM KCl, plus 500 μM adenine triphosphate (ATP) added fresh before use
  10. Protease inhibitors (1,000x stock solutions aliquoted and stored at -20 °C)
    10 μM leupeptin
    2 μM antipain
    1 mM benzamidine
    1 μg/ml pepstatin
    100 μM PMSF
    1 mM sodium orthovanadate
    1 mM dithiothreitol (DTT not used for GST-purifications)

Acknowledgments

This protocol was adapted as previously reported (Dunlop et al., 2009). This work was supported by an Association for International Cancer Research (AICR) career development award to AT and a Tuberous Sclerosis Association Junior Fellowship to KD.

References

  1. Bai, X., Ma, D., Liu, A., Shen, X., Wang, Q. J., Liu, Y. and Jiang, Y. (2007). Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38. Science 318(5852): 977-980.
  2. Burnett, P. E., Barrow, R. K., Cohen, N. A., Snyder, S. H. and Sabatini, D. M. (1998). RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci U S A 95(4): 1432-1437.
  3. Dodd, K. M., Yang, J., Shen, M. H., Sampson, J. R. and Tee, A. R. (2015). mTORC1 drives HIF-1alpha and VEGF-A signalling via multiple mechanisms involving 4E-BP1, S6K1 and STAT3. Oncogene 34(17): 2239-2250.
  4. Dunlop, E. A., Dodd, K. M., Seymour, L. A. and Tee, A. R. (2009). Mammalian target of rapamycin complex 1-mediated phosphorylation of eukaryotic initiation factor 4E-binding protein 1 requires multiple protein-protein interactions for substrate recognition. Cell Signal 21(7): 1073-1084.
  5. Kim, D. H., Sarbassov, D. D., Ali, S. M., King, J. E., Latek, R. R., Erdjument-Bromage, H., Tempst, P. and Sabatini, D. M. (2002). mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110(2): 163-175.
  6. Sancak, Y., Thoreen, C. C., Peterson, T. R., Lindquist, R. A., Kang, S. A., Spooner, E., Carr, S. A. and Sabatini, D. M. (2007). PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol Cell 25(6): 903-915.
  7. Schalm, S. S. and Blenis, J. (2002). Identification of a conserved motif required for mTOR signaling. Curr Biol 12(8): 632-639.

简介

历史上,使用温和的非离子去污剂如NP-40和或Triton-X100从哺乳动物细胞纯化雷帕霉素(mTOR)的机械目标,导致其天然激酶活性所必需的核心调节组分的解离。因此,这些较老的激酶测定需要MnCl 2以人工增强观察到的弱磷酸转移活性(Bai等人,2007; Kim等人)。 ,2002)。使用两性离子去污剂3 - [(3-胆碱酰氨基丙基)二甲基铵基] -1-丙磺酸盐(CHAPS),含有mTOR(Raptor)和Lst8(也称为GbetaL)的调节相关蛋白的mTOR复合物1(mTORC1)可以成功地纯化为复合物。这种体外激酶测定允许纯化类似于其生理状态并在生理性MgCl 2浓度下保持激酶活性的mTORC1(Sancak等人)。 ,2007)。 mTORC1的活性可以通过使用mTOR的激酶结构域内的过度活跃突变或包含补充到体外激酶测定中的富含脑部(Rheb)的GTP结合的RAS来进一步增强。 Rheb是结合并激活mTORC1以磷酸化下游底物的小G蛋白,例如真核起始因子4E-BP1(4E-BP1)(Burnett等人,1998),核糖体蛋白S6激酶1(S6K1)(Kim等人,2002),信号转导物和转录激活因子3(STAT3)(Dodd等人,2015)和脯氨酸富集的40kDa的Akt底物(PRAS40)(Dunlop等人,2009)。

材料和试剂

  1. HEK293E细胞
  2. 质粒和载体
    1. HA-Raptor(Addgene,目录号:8513)
    2. myc-mTOR(Addgene,目录号:1861)
    3. Rheb(国家生物技术信息中心,Gene,目录 编号:6009)使用网关克隆系统克隆到pDEST27中根据制造商方案(Life Technologies,目录 number:11812-013)
      注意:目前,它是"Thermo Fisher Scientific,Invitrogen™,目录号:11812-013"。
    4. 如先前所述产生的GST-4E-BP1/pGEX载体(Dunlop等人,2009)
  3. 抗体
    1. 克隆9E10抗-Myc抗体(Sigma-Aldrich,目录号:M5546)
    2. 克隆9B11抗-Myc抗体(Cell Signaling Technology,目录号:2276)
    3. 抗HA(Roche Diagnostics,目录号:11867431001)
    4. 抗GST(Merck Millipore Corporation,目录号:05-782)
  4. 细胞培养和转染
    1. Dulbecco改良的Eagle培养基(DMEM)
    2. 10%胎牛血清(FBS),EU Approved(South American)(Thermo Fisher Scientific,Gibco TM ,目录号:10270-106)
    3. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15070-063)
      注意:将HEK293E细胞在补充有10%FBS,1μg/ml青霉素和1μg/ml链霉素的DMEM中培养。
  5. 胰岛素(Sigma-Aldrich,目录号:I9278)
  6. 雷帕霉素(EMD Millipore公司,目录号:553210)
  7. 分析级别的化学品
    1. HEPES(Sigma-Aldrich,目录号:H3375)
    2. EDTA(Sigma-Aldrich,目录号:431788)
    3. (二钠盐,五水合物)(Sigma-Aldrich,目录号:50020)
    4. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
    5. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
    6. 腺嘌呤三磷酸(ATP)(Sigma-Aldrich,目录号:A26209)
    7. 亮肽素(Sigma-Aldrich,目录号:L5793)
    8. Antipain(Sigma-Aldrich,目录号:10791)
    9. 苯甲酰胺(Sigma-Aldrich,目录号:12072)
    10. 胃酶抑素A(Sigma-Aldrich,目录号:P5318)
    11. 钒酸钠(Sigma-Aldrich,目录号:289361)
    12. 二硫苏糖醇(Sigma-Aldrich,目录号:43815)
    13. 苯甲基磺酰氟(Sigma-Aldrich,目录号:78830)
    14. 渥曼青霉素(Sigma-Aldrich,目录号:W1628)
    15. 3 - [(3-胆酰氨基丙基)二甲基铵基] -1-丙磺酸盐水合物(CHAPS)(Sigma-Aldrich,目录号:226947)
  8. mTOR裂解缓冲液(参见配方)
  9. 低盐mTOR洗涤缓冲液(参见配方)
  10. 高盐mTOR洗涤缓冲液(参见配方)
  11. mTOR洗涤缓冲液(参见配方)
  12. 3x mTOR激酶测定缓冲液(参见配方)
  13. Rheb裂解缓冲液(参见配方)
  14. Rheb存储缓冲区(参见配方)
  15. 磷酸盐缓冲盐水(见配方)
  16. mTOR测定开始缓冲液(参见配方)
  17. 蛋白酶抑制剂(参见配方)

设备

  1. 热混合器加热块(Eppendorf AG,型号:Eppendorf thermomixer compact)
  2. 冷冻迷你离心机(Thermo Fisher Scientific,Thermo Scientific™,型号:Heraeus和Fresco 17离心机)
  3. Stuart TM TM SB2固定速度旋转器(Bibby Scientific Limited,Stuart Scientific)

程序

注意:整个过程的概述可以在图1中找到。



图1.mTOR 体外激酶测定如"方法"中所述,制备(A) mTORC1复合物,(B)Rheb-GTP和(D)药物抑制剂(雷帕霉素/FKBP12和渥曼青霉素)和mTORC1底物(4E-BP1)在mTORC1激酶测定(E)中

  1. 从HEK293E细胞产生mTOR/raptor复合物
    1. HEK293E细胞在Dulbecco改良的Eagle's培养基中培养 (DMEM),补充有10%胎牛血清(FBS)和1%,100μg/ml   青霉素和100μg/ml的链霉素
    2. 75cm 2烧瓶中的80% 汇合的HEK293E细胞用5μgMyc标记的共转染 mTOR和5μgHA标记的Raptor构建体或用a GST标记的Rheb构建体使用磷酸钙转染 方法(1×75cm 2的HEK293E细胞的烧瓶对于三种mTOR是足够的 激酶测定)。 转染后36小时收获细胞。 细胞是在裂解前用10μg/ml胰岛素处理30分钟。 这个剂量是 足以刺激mTORC1信号并确保活性 如先前所证实的,纯化复合物(Dunlop等人,2009)
    3. 刺激细胞与100 nM胰岛素15分钟,然后在1毫升 补充有蛋白酶抑制剂和0.3%CHAPS的mTOR裂解缓冲液 (w/v)。
    4. 在4℃下,以16,200×g离心8分钟(冷冻迷你离心机)。
    5. 孵育溶胞产物与3微升的Myc或HA抗体(mTORC1复杂   可以用HA- raptor或myc-mTOR免疫沉淀纯化) 在4℃下旋转1.5小时
    6. 使50%体积比的混合蛋白G加mTOR裂解缓冲液,每管加入40μl并孵育 在4℃下旋转(Stuart TM SB2固定速度旋转器)1小时。
    7. 用0.5ml补充有蛋白酶抑制剂的以下缓冲液洗涤免疫沉淀物:
      1. 1x低盐mTOR洗涤缓冲液(补充有0.3%w/v CHAPS)
      2. 2x高盐mTOR洗涤缓冲液(补充有0.3%w/v CHAPS)
      3. 2x mTOR洗涤缓冲液
    8. 分裂免疫沉淀等同地进入用于体外激酶测定的三个Eppendorf管中。

  2. 准备结合GTP的Rheb
    人类Rheb(基因ID:6009)克隆到含有N端GST标签的pDEST27中。
    1. 转染四个75cm 2的80%铺满的HEK293E细胞的烧瓶 与GST-Rheb-pDEST27(每瓶10μgDNA),使用标准钙 磷酸盐转染程序(Schalm等人,2002)。
    2. 在10%(v/v)FBS存在下培养HEK293E细胞过夜,直至完全融合(6-7×10 6个细胞)。
    3. 在补充有0.3%(w/v)CHAPS(加蛋白酶抑制剂)的1ml Rheb裂解缓冲液中裂解HEK293E细胞。
      注意:应省略还原剂(例如DTT),因为这将干扰 与GST-纯化。 孵育裂解物在冰上30分钟以利 裂解。
    4. 孵育预澄清的裂解物(在4℃下在16,200×g离心10分钟后)和固定的40μl50%体积比的混合物   Rheb裂解缓冲液和谷胱甘肽 - 琼脂糖珠,在4℃孵育2小时   °C旋转。
    5. 用0.5ml Rheb裂解缓冲液洗涤谷胱甘肽 - 琼脂糖珠两次   然后用0.5ml Rheb存储缓冲液补充一次 蛋白酶抑制剂。
    6. 从50μlRheb中的谷胱甘肽 - 琼脂糖珠上洗脱GST-Rheb 存储缓冲液补充有10mM谷胱甘肽(pH再调节 回到8.0)。
    7. 孵育洗脱GST-Rheb蛋白在30℃下10分钟与10毫米 EDTA和1mM GDP以产生无活性的Rheb-GDP或10mM EDTA和0.1 mM不可水解的GTPγS以产生活性Rheb-GTP。 紧密绑定 鸟嘌呤核苷酸至Rheb,添加MgCl 2至终浓度为20mM。 在冰上孵育直到使用。

  3. 产生用于阳性对照底物的去磷酸化4E-BP1蛋白质
    1. 用GST标记的4E-BP1/pGEX质粒转化BL21(DE3)pLys细菌 (4E-BP1 GeneID,1978)。
    2. 生长细菌直到OD 600是0.6-0.8,以诱导表达 加入异丙基-β-D-硫代半乳糖苷(IPTG)至终浓度并在30℃下孵育3小时。 通过离心沉淀细胞 在1,500×g下在4℃温育30分钟。
    3. 在补充有10mM EDTA,0.1%(v/v)Triton和蛋白酶抑制剂的10ml PBS中冻融细菌。
    4. 使用脉冲超声剪切细菌DNA [3 x 5秒周期全功率(30微米)]。 在4℃下以16,200×g离心10分钟,然后使用谷胱甘肽 - 琼脂糖珠从细菌上清液中纯化GST-4E-BP1。
    5. 脱磷酸GST-4E-BP1蛋白使用50 U虾碱 磷酸酶,在PBS,10mM EDTA,0.1%(v/v)Triton X-100中洗涤。 洗脱 在PBS(pH 7.6)中的10mM还原型谷胱甘肽中。
    6. 根据制造商协议使用HiTrap脱盐柱对洗脱液进行脱盐。
    7. 使用SDS-PAGE分离并用考马斯亮蓝染色检查 针对已知牛血清白蛋白(BSA)的纯度和浓度, 标准。
    8. 去磷酸化的4E-BP1可以在-80℃下以10μg/μl的等分试样保存以备将来使用。

  4. 准备FKBP12 /雷帕霉素药物/蛋白复合物抑制mTORC1
    1. 人FKBP12蛋白可以使用其表达和纯化 方案以产生上述GST-4E-BP1(省略去磷酸化 步骤C5)。
    2. FKBP12也可以在-80℃下以10μg/μl等分试样冷冻以备将来使用。
    3. 为了产生FKBP12 /雷帕霉素复合物:以10μl终体积(dH 2 O)。 在室温下在黑暗中孵育5分钟,并存储在冰上直到需要。

  5. 执行mTOR激酶测定
    1. 在3x mTOR激酶测定缓冲液中制备mTOR/Raptor免疫沉淀物 并根据需要加入75ng Rheb-GTP和/或2μlFKBP12 /雷帕霉素。
    2. 在开始激酶测定之前在冰上孵育20分钟。
    3. 加入10μlmTOR测定开始缓冲液,补充有500μMATP (在使用前新鲜加入)加150ng纯化的GST-4E-BP1或 测试底物以开始测定。 磷酸-4E-BP1(Thr 36/45)水平   用作阳性对照并指示mTOR激酶活性。
    4. 在30℃下在热混合器加热块中孵育30-60分钟,在20FCS下振荡。
    5. 通过加入4x样品缓冲液停止反应。
    6. 使用SDS-PAGE和western印迹分析样品。 代表性数据的示例如图2所示。

代表数据



图2.mTORC1 4E-BP1的定向磷酸化。在存在和不存在GTP的情况下进行的体外mTORC1激酶测定后,显示纯化的GST-4E-BP1的磷酸化的Western印迹 -Rheb。 myc-mTOR和HA-Raptor的水平显示为对照。

笔记

  1. 重要的是,Rheb从哺乳动物细胞而不是从细菌中纯化,因为其活性增强mTORC1需要正确的折叠和翻译后修饰(例如异戊烯化)。

食谱

注意:除非另有说明,否则所有缓冲液都储存在4°C。

  1. mTOR裂解缓冲液
    40mM HEPES(pH7.4) 2mM EDTA 10mMβ-甘油磷酸盐
  2. 低盐mTOR洗涤缓冲液
    40mM HEPES(pH7.4) 150mM NaCl 2mM EDTA 10mMβ-甘油磷酸盐
  3. 高盐mTOR洗涤缓冲液
    40mM HEPES(pH7.4) 400 mM NaCl
    2mM EDTA 10mMβ-甘油磷酸盐
  4. mTOR洗涤缓冲液
    25mM HEPES(pH7.4) 20 mM KCl
  5. 3x mTOR激酶测定缓冲液(等分并储存在-20℃) X3储备溶液:75mM HEPES(pH 7.4),60mM KCl,30mM MgCl 2
    将MgCl 2的0.5M储备液的1:50稀释液加入到25mM HEPES(pH 7.4),20mM KCl中。
  6. Rheb裂解缓冲液
    40mM HEPES(pH7.4) 10mM甘油磷酸盐5mM MgCl 2/
  7. Rheb存储缓冲区
    20mM HEPES(pH8.0) 200 mM NaCl
    5mM MgCl 2/
  8. 磷酸盐缓冲盐水
    137 mM NaCl 10 mM磷酸盐 2.7mM KCl(pH7.4)
  9. mTOR测定开始缓冲液(等分并保存在-20℃) 25mM HEPES(pH7.4) 10mM MgCl 2/
    140 mM KCl,以及500μM三磷酸腺苷(ATP),在使用前新鲜加入
  10. 蛋白酶抑制剂(1,000x储备溶液等分并储存在-20℃)
    10μM亮肽素
    2μM抗体
    1mM苄脒
    1μg/ml胃酶抑素
    100μMPMSF
    1mM原钒酸钠 1mM二硫苏糖醇(DTT不用于GST纯化)

致谢

该方案按照以前报道的方法进行改变(Dunlop等人,2009)。 这项工作是由国际癌症研究协会(AICR)职业发展奖向AT和结节性硬化症协会青年奖学金KD支持。

参考文献

  1. Bai,X.,Ma,D.,Liu,A.,Shen,X.,Wang,Q.J.,Liu,Y.and Jiang,Y。 Rheb通过拮抗其内源性抑制剂FKBP38激活mTOR。 em> 318(5852):977-980。
  2. Burnett,P.E.,Barrow,R.K.,Cohen,N.A.,Snyder,S.H。和Sabatini,D.M。(1998)。 翻译调节因子p70 S6激酶和4E-BP1的RAFT1磷酸化。 Proc Natl Acad Sci USA 95(4):1432-1437。
  3. Dodd,K.M.,Yang,J.,Shen,M.H.,Sampson,J.R.and Tee,A.R。(2015)。 mTORC1通过多种机制驱动HIF-1α和VEGF-A信号传导,包括4E-BP1,S6K1和STAT3 。 Oncogene 34(17):2239-2250。
  4. Dunlop,E.A.,Dodd,K.M.,Seymour,L.A。和Tee,A.R。(2009)。 雷帕霉素复合物1介导的真核起始因子4E结合蛋白1磷酸化的哺乳动物靶标需要多种蛋白 - 蛋白质相互作用用于底物识别。细胞信号21(7):1073-1084。
  5. Kim,D.H.,Sarbassov,D.D.,Ali,S.M.,King,J.E.,Latek,R.R.,Erdjument-Bromage,H.,Tempst,P.and Sabatini,D.M。(2002)。 mTOR与猛禽相互作用形成对细胞生长机械发出信号的营养敏感性复合物。 a> Cell 110(2):163-175。
  6. Sancak,Y.,Thoreen,C.C.,Peterson,T.R.,Lindquist,R.A.,Kang,S.A.,Spooner,E.,Carr,S.A。和Sabatini,D.M。(2007)。 PRAS40是mTORC1蛋白激酶的胰岛素调节抑制剂。 Mol 电池 25(6):903-915。
  7. Schalm,S.S。和Blenis,J。(2002)。 mTOR信号所需的保守基序的鉴定 Curr Biol (8):632-639。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Dodd, K. M. and Tee, A. R. (2016). In vitro mTORC1 Kinase Assay for Mammalian Cells Protocol. Bio-protocol 6(11): e1827. DOI: 10.21769/BioProtoc.1827.
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