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In vitro NLK Kinase Assay
体外NLK激酶测定   

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Abstract

This protocol provides step by step instructions to perform an in vitro kinase assay for nemo-like kinase. In addition, this protocol also describes an efficient method using mild lysis buffer for expression and purification of Glutathione S-transferase (GST) fusion proteins.

Keywords: NLK(NLK), in vitro kinase assay(体外激酶测定), GST-fusion protein(GST融合蛋白), YAP(YAP), Hippo pathway(Hippo通路)

Background

Drosophila nemo has been identified as a pivotal regulator of photoreceptor clusters in the developing Drosophila eyes (Choi and Benzer, 1994). It has been reported that nemo is conserved across various species. The mammalian homolog is nemo-like kinase (NLK) (Brott et al., 1998; Rocheleau et al., 1999). NLK is a member of the CMGC group (CDK, MAPK, GSK3 and CLK) and also belongs to the atypical MAPK family. NLK regulates multiple cellular mechanisms through transcription factors including TCF/LEF1, NICD, and FOXO, which are key players in diverse signaling pathways (Ishitani et al., 1999; Ishitani et al., 2010; Kim et al., 2010). Moreover, recently we have identified NLK as a novel kinase for Yes-associated protein (YAP), a co-transcriptional activator in the Hippo pathway (Moon et al., 2017). This protocol describes an in vitro method to measure the nemo-like kinase activity.

Materials and Reagents

  1. Pipette tips–1,000 μl tips (OHAUS, catalog number: PCB-1000 ), 200 μl tips (Neptune Scientific, catalog number: 2100 )
  2. 14 ml round bottom tubes (SPL, catalog number: 40014 )
  3. 15 ml conical centrifuge tubes (Ihanil Scientific, catalog number: LP-00015CP-00 )
  4. 60 mm cell culture dish (SPL, catalog number: 20060 )
  5. Microcentrifuge tube (SARSTEDT, catalog number: 72.690 )
  6. X-ray film cassette (Advansta, catalog number: L-07019-001 )
  7. X-ray film (XSANATEC, AGFA, catalog number: EA75Q )
  8. Cell scraper (SARSTEDT, catalog number: 83.1832 )
  9. Centrifuge bottles (Sigma-Aldrich, catalog number: B1033 )
  10. pGEX-4T-3 vector (GE Healthcare, catalog number: 28-9545-52 )
  11. pFLAG-CMV4 vector (Sigma-Aldrich, catalog number: E7158 )
  12. Flag-NLK vectors (Ishitani et al., 1999)
  13. Competent cells-BL21 (DE3) (EMD Millipore, Novagen, catalog number: 70235 )
  14. Homo sapiens (Human) HEK293 cells (ATCC, catalog number: CRL-1573 )
  15. Zinc chloride (ZnCl2) (Sigma-Aldrich, catalog number: 229997 )
  16. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Bio Basic, catalog number: IB0168 )
  17. Glutathione Sepharose 4B (GE Healthcare, catalog number: 17075601 )
  18. Ethanol (Merk, catalog number: 1.00983.1011 )
  19. Bovine serum albumin (BSA) (Bio Basic, catalog number: AD0023 )
  20. Dulbecco’s modified Eagle medium (DMEM) (Lonza, catalog number: 12-604F )
  21. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16000044 )
  22. Antibiotic-Antimycotic (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062 )
  23. TurboFect transfection reagents (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0531 )
  24. Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
  25. Anti-FLAG M2 antibody (Sigma-Aldrich, catalog number: F3165 )
  26. Protein A/G plus agarose (Santa Cruz Biotechnology, catalog number: sc-2003 )
  27. Adenosine 5’-triphosphate (ATP) [γ-32P] (PerkinElmer, catalog number: NEG502A )
  28. LB broth high salt (Duchefa Biochemie, catalog number: L1704 )
  29. Micro agar (Duchefa Biochemie, catalog number: M1002 )
  30. Ampicillin (Duchefa Biochemie, catalog number: A0104 )
  31. Tryptone (Duchefa Biochemie, catalog number: T1332 )
  32. Yeast extract (Duchefa Biochemie, catalog number: Y1333 )
  33. Sodium chloride (NaCl) (Duchefa Biochemie, catalog number: S0520 )
  34. Potassium chloride (KCl) (Duchefa Biochemie, catalog number: P0515 )
  35. Di-sodium hydrogen phosphate dihydrate (Na2HPO4·2H2O) (Duchefa Biochemie, catalog number: S0537 )
  36. Potassium phosphate dibasic (K2HPO4) (Merck, catalog number: 105108 )
  37. Tris (Duchefa Biochemie, catalog number: T1501 )
  38. NP-40 (IGEPAL CA-630) (Sigma-Aldrich, catalog number: I8896 )
  39. Ethylenediaminetetraacetic acid (EDTA) (Duchefa Biochemie, catalog number: E0511 )
  40. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’,-tetraacetic acid (EGTA) (MP Biomedicals, catalog number: 194823 )
  41. β-Glycerophosphate (MP Biomedicals, catalog number: 195206 )
  42. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S1504 )
  43. Dithiothreitol (DTT) (Bio Basic, catalog number: DB0058 )
  44. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  45. Leupeptin (Sigma-Aldrich, catalog number: L2884 )
  46. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  47. L-Glutathione reduced (Sigma-Aldrich, catalog number: G4251 )
  48. Methanol (Merck, catalog number: 106099 )
  49. Acetic acid (Merck, catalog number: 100056 )
  50. Coomassie Blue (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 20279 )
  51. HEPES (pH 7.5) (Duchefa Biochemie, catalog number: H1504.0100 )
  52. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
  53. Sodium dodecyl sulfate (SDS) (Duchefa Biochemie, catalog number: S1377 )
  54. Glycerol (Duchefa Biochemie, catalog number: G1345.1000 )
  55. β-Mercaptoethanol (AMRESCO, catalog number: 0482 )
  56. Bromophenol Blue (BPB) (AMRESCO, catalog number: 0449 )
  57. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
  58. Lysozyme (Agilent Technologies, catalog number: EC 3.2.1.17 )
  59. LB medium (see Recipes)
  60. LB agar plate (see Recipes)
  61. 2x YTA medium (see Recipes)
  62. 1x phosphate-buffered saline (PBS) (see Recipes)
  63. Cell lysis buffer A (see Recipes)
  64. Cell lysis buffer B (see Recipes)
  65. Elution buffer (see Recipes)
  66. Coomassie Blue staining buffer (see Recipes)
  67. 10x kinase buffer (see Recipes)
  68. 4x SDS loading dye (see Recipes)
  69. Destaining buffer (see Recipes)

Equipment

  1. Shaking incubator (VISION SCIENTIFIC, model: VS-8480SF )
  2. Erlenmeyer flask (Corning, PYREX®, catalog number: 4980-500 )
  3. UV spectrophotomter (Mecasys, Optizen, model: 2120UV )
  4. Centrifuge for 50/250 ml tubes
  5. 50 ml centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, model: 3119 )
  6. 250 ml centrifuge bottles (Thermo Fisher Scientific, Thermo ScientificTM, model: 3120 )
  7. Sonicator (Sonics & Materials, model: VCX 500 )
  8. High speed refrigerated micro centrifuge (TOMY DIGITAL BIOLOGY, model: MX-300 )
  9. Water-Jacketed CO2 incubator (Thermo Fisher Scientific, model: Forma Series II 3111 )
  10. Pipettes (Gilson, model: PIPETMAN®, catalog number: F167300 )
  11. Micro centrifuge rotor rack (TOMY DIGITAL BIOLOGY, model: AR015-24 )
  12. Micro centrifuge rotor rack (TOMY DIGITAL BIOLOGY, model: AR015-24 )
  13. Electrophoresis apparatus (Bio-Rad Laboratories, model: 1658033FC )
  14. Flat scanner (Epson, model: Epson Perfection V37 )
  15. Gel drying equipment (Vision Scientific, model: ME-2B-S )
  16. -80 °C freezer (NuAire, model: NU-6625D36 )
  17. Water bath (VISION SCIENTIFIC, model: VS-1205SW1 )
  18. Thermo shaker (BIOAND, model: MSC100 )
  19. Solid tapered microtip (Sonics & Materials, catalog number: 630-0418 )

Procedure

  1. Purification of GST-fusion protein
    1. Clone the gene of interest into the pGEX vector multiple cloning site.
    2. Transform the pGEX-YAP plasmid (Moon et al., 2017) into BL21 bacteria and then grow overnight on an LB plate with ampicillin (see Recipes).
      Note: The purification protocol can be applied to other GST fusion proteins after cloning the gene of interest in frame with GST into the multiple cloning site of the pGEX vector.
    3. Pick one colony and culture overnight at 37 °C in a shaking incubator (180 rpm) with 6 ml of 2x YTA medium in a 14 ml round bottom tube (add ampicillin; 300 μg/6 ml of 2x YTA medium, see Recipes).
    4. Transfer all cultured cells to Erlenmeyer flask.
    5. Incubate for 1 h at 37 °C in a shaking incubator (180 rpm) with 100 ml of 2x YTA (add ampicillin; 5 mg/100 ml of 2x YTA medium) and 50 μM ZnCl2.
    6. Measure optical density at 600 nm (OD600) by a UV spectrophotometer (OD should be between 0.5 and 0.8).
      Note: If OD600 is less than 0.5, incubate until it exceeds 0.5.
    7. To induce protein expression, add 1 ml of 100 mM IPTG to make a final concentration of 1 mM and incubate for 2 h at 37 °C in a shaking incubator.
      Note: Prepare 100 mM IPTG stock in sterile water; store at -20 °C.
    8. Centrifuge at 3,500 x g for 10 min at 4 °C and remove supernatant.
    9. Add 4 ml ice cold lysis buffer A (see Recipes) and resuspend the bacterial pellet by pipetting up and down followed by incubation for 10 min at 4 °C.
    10. Transfer the bacterial suspension to a 15 ml conical tube and add 1 mg of lysozyme followed by incubation for 20 min at 4 °C.
    11. Sonicate at output of 40% on ice for 10 sec (0.5 sec sonication/1 sec break) and keep on ice for 30 sec. Repeat sonication step one more time.
    12. Transfer lysate to four new 1.5 ml micro-centrifuge tubes (aliquot 1 ml) and then centrifuge at 14,000 x g for 10 min at 4 °C.

    Note: Henceforth, perform all procedures in micro-centrifuge tubes.

    1. Transfer supernatant to four new 1.5 ml micro-centrifuge tubes.
    2. Add 30 μl PBS-washed 50% glutathione Sepharose 4B slurry (see Notes) to each tube and then incubate while rotating for 90 min at 4 °C.
      Note: To remove the storage solution, wash 125 µl of the original glutathione Sepharose 4B slurry (it was stored in 20% ethanol) by adding with 1 ml ice cold PBS. Centrifuge at 500 x g for 5 min at 4 °C and discard supernatant. To obtain 50% glutathione Sepharose 4B slurry, resuspend the pellet in 100 µl 1x PBS.
    3. Centrifuge at 500 x g for 5 min at 4 °C and discard supernatant.
    4. Add 1 ml lysis buffer A (see Recipes) and then incubate while rotating for 10 min at 4 °C.
    5. Centrifuge at 500 x g for 5 min at 4 °C and discard supernatant.
    6. Wash by adding 1 ml of 50 mM Tris (pH 8.0) and immediately centrifuge at 500 x g for 5 min at 4 °C.
    7. Discard supernatant.
    8. Add 350 μl elution buffer (see Recipes) to the glutathione Sepharose 4B beads and incubate while rotating for 4 h or overnight at 4 °C.
    9. Centrifuge at 500 x g for 5 min at 4 °C and divide the supernatant into 20 µl aliquots and store the protein aliquots at -80 °C for future use.
    10. To quantify the amount of purified recombinant GST-YAP, run an aliquot of the purified protein with a BSA standard on a 10% SDS-PAGE. For this add 5 µl 4x SDS loading dye (see Recipes) to 15 µl sample and boil for 5 min.
      Note: Add 1x SDS loading dye to the other SDS-PAGE samples, e.g., molecular weight marker and BSA standard, to obtain a total volume of 20 μl.
    11. Separate 20 µl SDS-PAGE samples on 10 % SDS-PAGE.
    12. Run the gel for Coomassie Blue staining, then incubate the gel for 20 min at RT in Coomassie Blue staining buffer on a rocker, followed by 5 washes with destaining buffer, 10 min each time.
      Note: Quantify the final concentration and size of protein by Coomassie Blue staining with a BSA standard (see Figure 1 and Recipes).
    13. Store the aliquots at -80 °C freezer for future use.


      Figure 1. Coomassie Blue stained gel of purified GST-YAP. GST-YAP is evident as a 95 kDa band in the gel, GST alone runs at a molecular weight of around 25 kDa and BSA at 66 kDa. Lane M: protein size marker; Lane 1, 3, 5: GST-YAP; Lane 2, 4: GST; Lane 6: elution buffer; Lane 7: BSA (1 μg).

  2. In vitro NLK kinase assay
    1. Incubate HEK293 cells at 37 °C in a humidified 5% CO2 incubator in DMEM supplemented with 10% FBS and 1x antibiotics.
    2. Seed 0.8 x 106 HEK293 cells on two 60 mm untreated cell culture dishes.
    3. After 24 h, transfect cells with 3 μg Flag-NLK-WT (wild type) or Flag-NLK-KM (kinase negative) plasmid (Ishitani et al., 1999) according to the manufacturer’s protocol (TurboFect transfection reagent).
      Note: The ratio of DNA (μg) to TurboFect (μl) is 1:2 to 1:3.
    4. Wash cells once with 1 ml ice cold 1x PBS (see Recipes), harvest cells by scraping off the cells in ice cold 1 ml 1x PBS, transfer the cells to micro-centrifuge tubes and centrifuge at 14,000 x g for 5 min at 4 °C.
    5. Discard supernatant and add 200 μl lysis buffer B (see Recipes) to the pellet.
    6. Lyse the cells by pipetting up and down around 40 times with a 200 μl pipette and incubate for 30 min at 4 °C.
    7. Centrifuge at 14,000 x g for 5 min at 4 °C and transfer supernatant into new micro-centrifuge tubes.
    8. Measure protein concentration with Protein Assay Dye Reagent kit using UV spectrophotometer.
    9. Incubate 400 μg of protein in 800 μl of lysis buffer B with anti-FLAG antibody (0.5 μg) on rotator overnight at 4 °C.
    10. Add Protein A/G plus agarose (20 μl per sample).
    11. Incubate on microfuge-tube rotator for 120 min at 4 °C.
    12. Centrifuge at 3,000 x g for 2 min at 4 °C and discard supernatant.
    13. Add 800 μl of lysis buffer B (see Recipes) and incubate on rotator for 5 min at 4 °C.
    14. Centrifuge at 3,000 x g for 2 min at 4 °C and discard supernatant (repeat steps B12-B13 three times).
    15. Add and gently mix 800 μl of ice cold 1x kinase buffer (see Recipes) and immediately centrifuge at 3,000 x g for 2 min at 4 °C.
    16. Discard supernatant and add 3 μl of 10x kinase buffer, 1 μg GST-YAP (referred to section of ‘Purification of GST-fusion protein’), 10 μCi of Adenosine 5’-triphosphate (ATP) [γ-32P] and add H2O to make the total volume up to 30 μl in two different tubes (Flag-NLK-WT/-KM).
      Note: Radioactive isotopes should be used only by authorized persons and at authorized places.
    17. Incubate for 5 min at 25 °C.
    18. To stop the reaction, add 10 μl of 4x SDS loading dye (see Recipes) and boil for 5 min.
    19. Centrifuge at 3,000 x g for 2 min at 4 °C and carefully take 30 μl of supernatant and separate proteins on 10% SDS-PAGE.
    20. Detach SDS-PAGE gel from electrophoresis apparatus.
    21. Incubate gel at a rocker with Coomassie Blue staining buffer (see Recipes) at room temperature for 30 min.
    22. Discard staining buffer and rinse for 20 min with destaining buffer (see Recipes).
      Note: Repeat destaining until the background is nearly clear.
    23. Capture image of SDS-PAGE gel using a flat scanner for Coomassie Blue staining.
    24. Dry SDS-PAGE gel for 60 min in a Gel dryer.
    25. Expose radioactivity with X-ray film for 30 min to 4 h in X-ray film cassette in a dark room (Figure 2).
    26. Develop and fix X-ray film for analyzing NLK kinase activity (see Data analysis).


      Figure 2. Schematic diagram of gel exposure

Data analysis

We have performed the in vitro kinase assay by incubating GST-YAP with immunoprecipitated Flag-NLK constructs (wild type or kinase negative) from lysates of HEK293 cells transfected with Flag-NLK. The level of autoradiography was detected by Flag-NLK-WT, but Flag-NLK-KM was not evident (Figure 3, red box). The in vitro NLK Kinase Assay suggests that NLK interacts with and directly phosphorylates YAP. Note that image analysis software (ImageJ) can be used to quantify the intensity of radiography. For this, we compared the band intensities of the phosphorylated GST-YAP (autoradiography) normalized to the total amount of GST-YAP (Coomassie Blue staining).


Figure 3. NLK phosphorylates YAP in vitro. HEK293 cells were transiently transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag antibodies and in vitro kinase assays were performed. The phosphorylation of GST-YAP and autophosphorylation of NLK were shown by autoradiography (top panel). Similar levels of GST-YAP and Flag-NLK were visualized by Coomassie Blue staining (bottom panel). This figure has been modified from Moon et al. (2017). EMBO Rep. doi:10.15252/embr.201642683

Recipes

  1. LB medium (autoclaved, store at 4 °C)
    25 g/L LB broth high salt
  2. LB agar plate (store at 4 °C)
    25 g/L LB broth high salt
    15 g/L Micro agar
    Autoclave and pour it into plates. Cool down in sterile place
  3. 2x YTA medium (autoclaved, store at 4 °C)
    16 g/L tryptone
    10 g/L yeast extract
    5 g/L NaCl
    Adjust pH to 7.0
  4. 1x phosphate-buffered saline (PBS) (autoclaved, store at 4 °C)
    8 g/L NaCl
    0.2 g/L KCl
    1.46 g/L Na2HPO4
    0.24 g/L KH2PO4
    Adjust pH to 7.4
  5. Cell lysis buffer A (store at -20 °C)
    50 mM Tris (pH 7.5)
    150 mM NaCl
    0.05% NP-40 (IGEPAL CA-630)
  6. Cell lysis buffer B (prepare daily fresh, store at 4 °C)
    20 mM Tris (pH 7.5)
    100 mM NaCl
    1 mM EDTA
    2 mM EGTA
    50 mM β-glycerophosphate
    50 mM NaF
    1 mM sodium vanadate
    2 mM dithiothreitol (DTT)
    1 mM phenylmethylsulfonyl fluoride (PMSF)
    1 μg/ml of leupeptin
    1% Triton X-100
  7. Elution buffer (prepare daily fresh)
    50 mM Tris (pH 8.0)
    6.8 mg/ml reduced glutathione
  8. Coomassie Blue staining buffer (store at RT)
    450 ml/L methanol
    100 ml/L acetic acid
    2.5 g/L Coomassie Blue
  9. 10x kinase buffer (store at -20 °C)
    100 mM HEPES (pH 7.5)
    10 mM dithiothreitol (DTT)
    50 mM MgCl2
  10. 4x SDS loading dye (store at -20 °C)
    200 mM Tris (pH 6.8)
    5% SDS
    25% glycerol
    200 mM β-mercaptoethanol
    0.4% bromophenol blue (BPB)
  11. Destaining buffer (store at RT)
    450 ml/L methanol
    100 ml/L acetic acid

Acknowledgments

This protocol was adapted from the research article by Moon et al. (2017). FLAG-NLK constructs were kindly provided by Dr. Ishitani T from Kyushu University, Japan. This work was supported by the grants from the National Research Foundation of Korea (2016R1E1A1A01943544) to E. Jho. There is no conflict of interest or competing interest that may impact the design and implementation of their protocol.

References

  1. Brott, B. K., Pinsky, B. A. and Erikson, R. L. (1998). Nlk is a murine protein kinase related to Erk/MAP kinases and localized in the nucleus. Proc Natl Acad Sci U S A 95(3): 963-968.
  2. Choi, K. W. and Benzer, S. (1994). Rotation of photoreceptor clusters in the developing Drosophila eye requires the nemo gene. Cell 78(1): 125-136.
  3. Ishitani, T., Hirao, T., Suzuki, M., Isoda, M., Ishitani, S., Harigaya, K., Kitagawa, M., Matsumoto, K. and Itoh, M. (2010). Nemo-like kinase suppresses Notch signalling by interfering with formation of the Notch active transcriptional complex. Nat Cell Biol 12(3): 278-285.
  4. Ishitani, T., Ninomiya-Tsuji, J., Nagai, S., Nishita, M., Meneghini, M., Barker, N., Waterman, M., Bowerman, B., Clevers, H., Shibuya, H. and Matsumoto, K. (1999). The TAK1-NLK-MAPK-related pathway antagonizes signalling between β-catenin and transcription factor TCF. Nature 399(6738): 798-802.
  5. Kim, S., Kim, Y., Lee, J. and Chung, J. (2010). Regulation of FOXO1 by TAK1-Nemo-like kinase pathway. J Biol Chem 285(11): 8122-8129.
  6. Moon, S., Kim, W., Kim, S., Kim, Y., Song, Y., Bilousov, O., Kim, J., Lee, T., Cha, B., Kim, M., Kim, H., Katanaev, V. L. and Jho, E. H. (2017). Phosphorylation by NLK inhibits YAP-14-3-3-interactions and induces its nuclear localization. EMBO Rep 18(1): 61-71.
  7. Rocheleau, C. E., Yasuda, J., Shin, T. H., Lin, R., Sawa, H., Okano, H., Priess, J. R., Davis, R. J. and Mello, C. C. (1999). WRM-1 activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals in C. elegans. Cell 97(6): 717-726.

简介

该协议提供了一步一步的指导,以进行nemo样激酶的体外激酶测定。 此外,该方案还描述了使用温和裂解缓冲液来表达和纯化谷胱甘肽S-转移酶(GST)融合蛋白的有效方法。

【背景】果蝇nemo已经被确定为在发育中的果蝇眼中光感受器簇的关键调节剂(Choi和Benzer,1994)。 据报道,nemo是不同物种间保守的。 该哺乳动物同系物是类似于Nemo的激酶(NLK)(Brott等人,1998; Rocheleau等人,1999)。 NLK是CMGC集团(CDK,MAPK,GSK3和CLK)的成员,也属于非典型的MAPK家族。 NLK通过转录因子调节多种细胞机制,所述转录因子包括在不同信号传导途径中的关键参与者TCF / LEF1,NICD和FOXO(Ishitani等人,1999; Ishitani等人, ,2010; Kim等人,2010)。 此外,最近我们已经将NLK鉴定为Yes相关蛋白(YAP)的新型激酶,YAP是Hippo途径中的共转录激活剂(Moon等人,2017)。 该协议描述了用于测量类似尼莫地来激酶活性的体外方法。

关键字:NLK, 体外激酶测定, GST融合蛋白, YAP, Hippo通路

材料和试剂

  1. 移液器吸头 - 1000μl吸头(OHAUS,产品目录号:PCB-1000),200μl吸头(Neptune Scientific,目录号:2100)

  2. 14毫升圆底管(SPL,目录号:40014)
  3. 15ml锥形离心管(Ihanil Scientific,目录号:LP-00015CP-00)
  4. 60毫米细胞培养皿(SPL,目录号:20060)
  5. 微量离心管(SARSTEDT,目录号:72.690)
  6. X光胶片暗盒(Advansta,目录号:L-07019-001)
  7. X光片(XSANATEC,AGFA,目录号:EA75Q)
  8. 细胞刮刀(SARSTEDT,目录号:83.1832)
  9. 离心瓶(Sigma-Aldrich,目录号:B1033)
  10. pGEX-4T-3载体(GE Healthcare,目录号:28-9545-52)
  11. pFLAG-CMV4载体(Sigma-Aldrich,目录号:E7158)
  12. Flag-NLK载体(Ishitani等人,1999)
  13. 感受态细胞-BL21(DE3)(EMD Millipore,Novagen,目录号:70235)
  14. (人)HEK293细胞(ATCC,目录号:CRL-1573)
  15. 氯化锌(ZnCl 2)(Sigma-Aldrich,目录号:229997)
  16. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Bio Basic,目录号:IB0168)
  17. 谷胱甘肽琼脂糖4B(GE Healthcare,目录号:17075601)
  18. 乙醇(Merk,目录号:1.00983.1011)
  19. 牛血清白蛋白(BSA)(Bio Basic,目录号:AD0023)
  20. 达尔伯克改良伊格尔培养基(DMEM)(Lonza,目录号:12-604F)
  21. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:16000044)
  22. 抗生素 - 抗真菌药(赛默飞世尔科技(Thermo Fisher Scientific),Gibco TM,目录号:15240062)
  23. TurboFect转染试剂(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0531)
  24. 蛋白质分析染料试剂浓缩物(Bio-Rad Laboratories,目录号:5000006)
  25. 抗FLAG M2抗体(Sigma-Aldrich,目录号:F3165)
  26. 蛋白A / G加琼脂糖(Santa Cruz Biotechnology,目录号:sc-2003)
  27. 腺苷5'-三磷酸(ATP)[γ-32 P](PerkinElmer,目录号:NEG502A)
  28. LB肉汤高盐(Duchefa Biochemie,目录号:L1704)
  29. 微琼脂(Duchefa Biochemie,目录号:M1002)
  30. 氨苄青霉素(Duchefa Biochemie,目录号:A0104)
  31. 胰蛋白胨(Duchefa Biochemie,目录号:T1332)
  32. 酵母提取物(Duchefa Biochemie,目录编号:Y1333)
  33. 氯化钠(NaCl)(Duchefa Biochemie,目录号:S0520)
  34. 氯化钾(KCl)(Duchefa Biochemie,目录号:P0515)
  35. 磷酸二氢钠二水合物(Na 2 HPO 4•2H 2 O)(Duchefa Biochemie,目录号:S0537)
  36. 磷酸二氢钾(K 2 HPO 4)(Merck,目录号:105108)
  37. Tris(Duchefa Biochemie,目录号:T1501)
  38. NP-40(IGEPAL CA-630)(Sigma-Aldrich,目录号:I8896)
  39. 乙二胺四乙酸(EDTA)(Duchefa Biochemie,目录号:E0511)
  40. 乙二醇 - 双(2-氨基乙醚) - N,N,N',N', - 四乙酸(EGTA)(MP Biomedicals,目录号:194823)
  41. β-甘油磷酸酯(MP Biomedicals,目录号:195206)
  42. 氟化钠(NaF)(Sigma-Aldrich,目录号:S1504)
  43. 二硫苏糖醇(DTT)(Bio Basic,目录号:DB0058)
  44. 苯基甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  45. 亮肽素(Sigma-Aldrich,目录号:L2884)
  46. Triton X-100(Sigma-Aldrich,目录号:T8787)
  47. L-谷胱甘肽减少(Sigma-Aldrich,目录号:G4251)
  48. 甲醇(Merck,目录号:106099)
  49. 醋酸(Merck,目录号:100056)
  50. 考马斯蓝(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:20279)
  51. HEPES(pH 7.5)(Duchefa Biochemie,目录号:H1504.0100)
  52. 氯化镁六水合物(MgCl 2•6H 2 O)(Sigma-Aldrich,目录号:M2670)
  53. 十二烷基硫酸钠(SDS)(Duchefa Biochemie,目录号:S1377)
  54. 甘油(Duchefa Biochemie,目录号:G1345.1000)
  55. β-巯基乙醇(AMRESCO,目录号:0482)
  56. 溴酚蓝(BPB)(AMRESCO,目录号:0449)
  57. 原钒酸钠(Sigma-Aldrich,目录号:S6508)
  58. 溶菌酶(安捷伦科技公司,产品目录号:EC 3.2.1.17)
  59. LB培养基(见食谱)
  60. LB琼脂平板(见食谱)
  61. 2x YTA中(见食谱)
  62. 1x磷酸盐缓冲盐水(PBS)(见食谱)
  63. 细胞溶解缓冲液A(见食谱)
  64. 细胞溶解缓冲液B(见食谱)
  65. 洗脱缓冲液(见食谱)
  66. 考马斯蓝染色缓冲液(见食谱)
  67. 10倍激酶缓冲液(见食谱)
  68. 4倍SDS加载染料(见食谱)
  69. 去沉淀缓冲液(见食谱)

设备

  1. 摇动培养箱(VISION SCIENTIFIC,型号:VS-8480SF)
  2. 锥形瓶(Corning,PYREX®,目录号:4980-500)
  3. 紫外分光光度计(Mecasys,Optizen,型号:2120UV)
  4. 离心50/250毫升管
  5. 50ml离心管(Thermo Fisher Scientific,Thermo Scientific TM,型号:3119)
  6. 250ml离心瓶(Thermo Fisher Scientific,Thermo Scientific TM,型号:3120)
  7. 超声波仪(Sonics& Materials,型号:VCX 500)
  8. 高速冷冻微量离心机(TOMY DIGITAL BIOLOGY,型号:MX-300)
  9. 水包裹CO 2培养箱(Thermo Fisher Scientific,型号:Forma Series II 3111)
  10. 移液器(Gilson,型号:PIPETMAN®,,产品目录号:F167300)
  11. 微型离心机转子架(TOMY DIGITAL BIOLOGY,型号:AR015-24)
  12. 微型离心机转子架(TOMY DIGITAL BIOLOGY,型号:AR015-24)
  13. 电泳装置(Bio-Rad Laboratories,型号:1658033FC)
  14. 平板扫描仪(爱普生,型号:爱普生Perfection V37)
  15. 凝胶干燥设备(Vision Scientific,型号:ME-2B-S)
  16. -80°C冰柜(NuAire,型号:NU-6625D36)
  17. 水浴(VISION SCIENTIFIC,型号:VS-1205SW1)
  18. 热摇床(BIOAND,型号:MSC100)
  19. 实心锥形微尖端(Sonics& Materials,目录号:630-0418)

程序

  1. GST融合蛋白的纯化
    1. 将感兴趣的基因克隆到pGEX载体多克隆位点。
    2. 将pGEX-YAP质粒(Moon等人,2017)转化到BL21细菌中,然后在含有氨苄青霉素的LB平板上生长过夜(参见食谱)。
      注意:将目的基因与GST一致克隆到pGEX载体的多克隆位点后,可以将纯化方案应用于其他GST融合蛋白。
    3. 挑取一个菌落,并在14ml圆底试管(加氨苄青霉素;300μg/ 6ml 2x YTA培养基,参见食谱)中用6ml 2x YTA培养基在振荡培养箱(180rpm)中于37℃培养过夜。
    4. 将所有培养的细胞转移到锥形瓶中。
    5. 在37℃,振荡培养箱(180转每分钟)中用100ml 2×YTA(加入氨苄青霉素; 5mg / 100ml 2×YTA培养基)和50μMZnCl 2孵育1小时。 br />
    6. 通过紫外分光光度计测量600nm(OD 600)的光密度(OD应该在0.5和0.8之间)。
      小于0.5,孵化至超过0.5。
    7. 为了诱导蛋白质表达,加入1ml 100mM IPTG以使最终浓度为1mM,并在37℃下在摇动培养箱中温育2小时。
      注意:在无菌水中准备100mM IPTG原液;存放在-20°C。

    8. 在3,500 xg g离心10分钟在4°C和去除上清液。
    9. 加入4毫升冰冷裂解缓冲液A(见食谱),并通过上下移液重悬细菌沉淀,然后在4℃孵育10分钟。
    10. 将细菌悬浮液转移到15ml锥形管中,加入1mg溶菌酶,然后在4℃孵育20分钟。
    11. 在冰上40%的输出超声10秒(超声0.5秒/中断1秒),冰上30秒。再次重复超声波步骤。
    12. 将溶胞产物转移至四个新的1.5ml微量离心管(等分试样1ml)中,然后在4℃以14,000xg离心10分钟。

    注意:今后,在微量离心管中进行所有操作。

    1. 转移上清到四个新的1.5毫升微型离心管。
    2. 添加30μLPBS洗过的50%谷胱甘肽琼脂糖4B浆液(见注释),然后孵化,同时在4°C旋转90分钟。
      注意:为了去除储存液,加入1 ml冰冷的PBS洗125μl原始的谷胱甘肽琼脂糖4B浆液(储存在20%乙醇中)。在4℃以500×g离心5分钟并弃去上清液。为了获得50%的谷胱甘肽琼脂糖4B浆液,将沉淀重悬于100μl1x PBS中。

    3. 在4℃500×g离心5分钟,弃去上清液。
    4. 加入1 ml裂解缓冲液A(见食谱),然后在4℃下旋转10分钟进行孵育。

    5. 在4℃500×g离心5分钟,弃去上清液。
    6. 通过加入1ml 50mM Tris(pH8.0)进行洗涤并立即在4℃以500gxg离心5分钟。
    7. 丢弃上清。
    8. 添加350μL的洗脱缓冲液(见食谱)谷胱甘肽琼脂糖4B珠和孵化,同时旋转4小时或在4°C过夜。
    9. 在500℃下离心5分钟5分钟,并将上清液分成20μl等分试样,并将蛋白等分试样储存在-80℃以备将来使用。
    10. 为了定量纯化的重组GST-YAP的量,在10%SDS-PAGE上用BSA标准品运行等分的纯化蛋白质。为此,加入5μl4x SDS上样染料(见食谱)至15μl样品并煮沸5分钟。
      注意:将1x SDS上样染料加入到其他SDS-PAGE样品中,例如分子量标记和BSA标准,以获得20μl的总体积。

    11. 在10%SDS-PAGE上分离20μlSDS-PAGE样品
    12. 运行凝胶进行考马斯蓝染色,然后在摇床上的考马斯蓝染色缓冲液中室温孵育20分钟,然后用脱色缓冲液洗5次,每次10分钟。
      注意:用BSA标准品通过考马斯蓝染色来定量蛋白质的终浓度和大小(见图1和配方)。
    13. 将等分试样储存在-80°C冰箱中备用。


      图1.纯化的GST-YAP的考马斯蓝染色的凝胶GST-YAP在凝胶中显示为95kDa的条带,单独的GST以25kDa的分子量运行,66kDa的BSA 。泳道M:蛋白质大小标记;泳道1,3,5:GST-YAP;泳道2,4:GST;泳道6:洗脱缓冲液;泳道7:BSA(1微克)。

  2. 在体外 NLK激酶测定
    1. 在补充有10%FBS和1x抗生素的DMEM中的湿润的5%CO 2培养箱中37°C孵育HEK293细胞。
    2. 在两个60mm的未处理的细胞培养皿中播种0.8×10 6个HEK293细胞。
    3. 24小时后,根据制造商的方案(3μg)使用3μgFlag-NLK-WT(野生型)或Flag-NLK-KM(激酶阴性)质粒(Ishitani等人,1999)转染细胞TurboFect转染试剂)。
      注:DNA(μg)与TurboFect(μl)的比例为1:2至1:3。
    4. 用1ml冰冷的1x PBS洗涤细胞一次(参见食谱),通过在冰冷的1ml 1x PBS中刮下细胞收获细胞,将细胞转移到微量离心管中并以14,000xg /在4°C 5分钟。
    5. 弃去上清液,加入200μl裂解缓冲液B(见食谱)到沉淀。
    6. 用200μl移液管上下移液约40次,溶解细胞,在4℃孵育30分钟。
    7. 在14,000×g离心5分钟,并将上清液转移到新的微量离心管中。
    8. 使用蛋白质分析染料试剂盒使用紫外分光光度计测量蛋白质浓度。
    9. 在800μl裂解缓冲液B中用400μl转染蛋白上的抗FLAG抗体(0.5μg)在4℃孵育过夜。
    10. 添加蛋白A / G加琼脂糖(每个样品20μl)。

    11. 在微型离心管转子上4℃孵育120分钟
    12. 在4℃3000×g离心2分钟,弃去上清液。
    13. 加入800μl的裂解缓冲液B(见食谱),并在4℃下旋转孵育5分钟。

    14. 在4℃3000×g离心2分钟并弃去上清液(重复步骤B12-B13三次)。
    15. 加入并轻轻混合800μl冰冷的1x激酶缓冲液(参见食谱),立即在4℃下以3,000gxg离心2分钟。
    16. 弃去上清,加入3μl10x激酶缓冲液,1μgGST-YAP(参见“纯化GST融合蛋白”一节),10μCi腺苷5'-三磷酸(ATP)[γ- (Flag-NLK-WT / -KM),并加入H 2 O使总体积达到30μl(Flag-NLK-WT / -KM)。
      注:放射性同位素只能由授权人员在授权的地方使用。

    17. 在25°C孵育5分钟
    18. 停止反应,加入10微升4倍SDS加载染料(见食谱)并煮沸5分钟。
    19. 在4℃3000×g离心2分钟,小心取30μl上清液,在10%SDS-PAGE上分离蛋白质。

    20. 从电泳仪上分离SDS-PAGE凝胶

    21. 在室温下用考马斯蓝染色缓冲液(见食谱)在摇杆上孵育凝胶30分钟。
    22. 弃去染色缓冲液,用去污缓冲液冲洗20分钟(见食谱)。
      注意:重复脱色直到背景几乎清晰。

    23. 使用平板扫描仪捕获SDS-PAGE凝胶图像进行考马斯蓝染色
    24. 在凝胶干燥器中干燥SDS-PAGE凝胶60分钟。

    25. 在黑暗的房间里用X光胶片暗箱放射X线胶片放射30分钟到4小时。
    26. 开发和修复X射线胶片分析NLK激酶活性(见数据分析)。


      图2.凝胶暴露的示意图

数据分析

我们已经通过用来自用Flag-NLK转染的HEK293细胞的裂解物孵育GST-YAP与免疫沉淀的Flag-NLK构建体(野生型或激酶阴性)来进行体外激酶测定。 Flag-NLK-WT检测到放射自显影的水平,但Flag-NLK-KM不明显(图3,红框)。体外 NLK激酶分析表明NLK与YAP相互作用并直接磷酸化YAP。请注意,图像分析软件( ImageJ )可用于量化放射摄影的强度。为此,我们比较了磷酸化的GST-YAP(放射自显影)与GST-YAP(考马斯蓝染色)总量的标准化带的强度。


图3. NLK体外磷酸化YAP 用指定的质粒瞬时转染HEK293细胞。用抗Flag抗体免疫沉淀细胞裂解物,并进行体外激酶测定。放射自显影显示GST-YAP的磷酸化和NLK的自磷酸化(上图)。通过考马斯亮蓝染色可见相似水平的GST-YAP和Flag-NLK(下图)。这个数字已经从Moon et。(2017)进行了修改。 EMBO Rep。doi:10.15252 / embr.201642683

食谱

  1. LB培养基(高压灭菌,4°C储存)
    25克/升LB肉汤高盐
  2. LB琼脂平板(4°C储存)
    25克/升LB肉汤高盐
    15克/升微型琼脂
    高压灭菌器倒入板中。在无菌的地方冷静下来
  3. 2x YTA培养基(高压灭菌,4°C储存)
    16克/升胰蛋白胨
    10克/升酵母提取物
    5克/升NaCl
    调整pH值到7.0
  4. 1x磷酸盐缓冲液(PBS)(高压灭菌,4°C储存)
    8克/升NaCl
    0.2克/升KCl
    1.46 g / L Na 2 HPO 4 4 0.24g / L KH 2 PO 4 4 调整pH值到7.4
  5. 细胞裂解缓冲液A(在-20°C储存)
    50 mM Tris(pH 7.5)
    150 mM NaCl
    0.05%NP-40(IGEPAL CA-630)
  6. 细胞裂解缓冲液B(每天新鲜制备,储存在4°C)
    20 mM Tris(pH 7.5)
    100 mM NaCl
    1 mM EDTA
    2 mM EGTA
    50毫克β-甘油磷酸酯
    50 mM NaF
    1 mM钒酸钠
    2 mM二硫苏糖醇(DTT)
    1毫米苯甲基磺酰氟(PMSF)
    1μg/ ml亮肽素
    1%Triton X-100
  7. 洗脱缓冲液(每日新鲜)
    50 mM Tris(pH 8.0)
    6.8毫克/毫升还原型谷胱甘肽
  8. 考马斯蓝染色缓冲液(保存在RT)
    450毫升/升甲醇
    100毫升/升乙酸
    2.5克/升考马斯蓝
  9. 10倍激酶缓冲液(在-20°C储存)
    100毫米HEPES(pH 7.5)
    10 mM二硫苏糖醇(DTT)
    50mM MgCl 2 2/2
  10. 4倍SDS加载染料(在-20°C储存)
    200 mM Tris(pH 6.8)
    5%SDS
    25%甘油
    200 mMβ-巯基乙醇
    0.4%溴酚蓝(BPB)
  11. 暂存缓冲区(在RT存储)
    450毫升/升甲醇
    100毫升/升乙酸

致谢

该协议是根据Moon等人的研究文章改编的。 FLAG-NLK构建体由日本九州大学的Ishitani T博士提供。这项工作得到了韩国国家研究基金会(2016R1E1A1A01943544)对E.Jho的支持。没有可能影响其协议设计和实施的利益冲突或利益冲突。

参考

  1. Brott,B.K.,Pinsky,B.A。和Erikson,R.L。(1998)。 Nlk是一种与Erk / MAP激酶相关的小鼠蛋白激酶,位于细胞核内。
    Proc Natl Acad Sci U S A 95(3):963-968。
  2. Choi,K.W。和Benzer,S。(1994)。 在发展中的果蝇眼中旋转感光团需要nemo基因。 细胞 78(1):125-136。
  3. Ishitani,T.,Hirao,T.,Suzuki,M.,Isoda,M.,Ishitani,S.,Harigaya,K.,Kitagawa,M.,Matsumoto,K。和Itoh,M。(2010)。 Nemo样激酶通过干扰Notch活性转录复合物的形成来抑制Notch信号。 Nat Cell Biol 12(3):278-285。
  4. Ishitani,T.,Ninomiya-Tsuji,J.,Nagai,S.,Nishita,M.,Meneghini,M.,Barker,N.,Waterman,M.,Bowerman,B.,Clevers,H.,Shibuya,H 。和Matsumoto,K。(1999)。 TAK1-NLK-MAPK相关途径对抗β-连环蛋白和转录因子TCF之间的信号传导。 /> Nature 399(6738):798-802。
  5. Kim,S.,Kim,Y.,Lee,J。和Chung,J。(2010)。 TAK1-Nemo样激酶途径对FOXO1的调节 J Biol Chem 285(11):8122-8129。
  6. Moon,S.,Kim,W.,Kim,S.,Kim,Y.,Song,Y.,Bilousov,O.,Kim,J.,Lee,T.,Cha,B.,Kim, Kim,H.,Katanaev,VL和Jho,EH(2017)。 NLK磷酸化抑制YAP-14-3-3相互作用并诱导其核定位。 a> EMBO Rep 18(1):61-71。
  7. Rocheleau,C.E.,Yasuda,J.,Shin,T.H.,Lin,R.,Sawa,H.,Okano,H.,Priess,J.R。,Davis,R.J。和Mello,C.C。(1999)。 WRM-1激活LIT-1蛋白激酶以转导的前/后极性信号, C。 elegans 。 Cell 97(6):717-726。
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Moon, S., Kim, J. and Jho, E. (2017). In vitro NLK Kinase Assay. Bio-protocol 7(21): e2593. DOI: 10.21769/BioProtoc.2593.
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