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ARP2/3 Phosphorylation Assay in the Presence of Recombinant Bacterial Effectors
重组细菌效应子存在条件下ARP2/3磷酸化修饰的检测   

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Abstract

The Actin-Related Protein 2/3 (ARP2/3) complex is an actin nucleator that generates a branched actin network in mammalian cells. In addition to binding nucleation promoting factors, LeClaire et al. demonstrated that its phosphorylation state is essential key for its activity (LeClaire et al., 2008). In cells, the ARP2/3 complex is phosphorylated on threonine and tyrosine residues of the ARP2, ARP3, and ARPC1 subunits (Vadlamudi et al., 2004; LeClaire et al., 2008; Narayanan et al., 2011; LeClaire et al., 2015). In particular, phosphorylation of threonine 237 and 238 of the ARP2 subunit is necessary to allow a change in the ARP2/3 complex structure to its active conformation (Narayanan et al., 2011; LeClaire et al., 2015). While important for many functions in eukaryotic cells, ARP2/3 complex activity also benefits several cellular pathogens (Haglund and Welch, 2011; Welch and Way, 2013). Recently, we demonstrated that the bacterial pathogen, Legionella pneumophila, manipulates ARP2/3 complex phosphorylation state using a bacterial protein kinase injected in host cell cytoplasm (Michard et al., 2015). Here, we describe how to test the ability of a bacterial protein kinase or another protein kinase to phosphorylate the ARP2/3 complex in an in vitro context. First, the ARP2/3 complex and the bacterial protein kinase are produced and purified. Then, the purified proteins are incubated in the presence of ATP, and the ARP2/3 phosphorylation level is analyzed by Western blot.

Keywords: in vitro phosphorylation assay(体外磷酸化测定), Protein kinase(蛋白激酶), ARP2/3 complex(ARP2/3复合物), Protein purification(蛋白质纯化), Western blot analysis(蛋白质印迹分析)

Background

The ARP2/3 complex is phosphorylated on threonine and tyrosine residues (LeClaire et al., 2008). Four phosphorylation sites on the ARPC1 and ARP2 subunits of ARP2/3 complex are currently known: the threonine 21 of ARPC1, threonines 237/238 and tyrosine 202 of ARP2, each demonstrating an important role for ARP2/3 function (Vadlamudi et al., 2004; LeClaire et al., 2008; Narayanan et al., 2011). A recent study by our laboratory demonstrated that the Legionella kinase 2 (LegK2), an effector serine/threonine protein kinase of Legionella pneumophila, modifies the threonine phosphorylation state of ARPC1B and ARP3 subunits of ARP2/3 complex. ARP2/3 complex phosphorylation inactivates and blocks actin polymerization on the bacterial vacuole, preventing the degradation of bacteria by the endocytic pathway (Michard et al., 2015). This protocol describes an in vitro phosphorylation assay routinely used in our laboratory to test a potential substrate of protein kinases as subunits of the ARP2/3 complex. The protocol can be also adapted to assay other substrates as needed.

Materials and Reagents

  1. Overproduction and extraction of bacterial protein kinase
    1. 50 ml conical tubes (Greiner Bio One International, catalog number: 227261 )
    2. Sterile culture tubes (SARSTEDT, catalog number: 62.515.006 )
    3. Sterilization filters (0.22 µm) (Dutscher Scientific, catalog number: 51732 )
    4. Spectrophotometer cuvette (Dutscher Scientific, catalog number: 030101 )
    5. Escherichia coli BL21 (pREP4-groESL) (Amrein et al., 1995)
    6. pGEX-6P-3 (Smith and Johnson, 1988)/pGEX-protein kinase
    7. Prechilled dH2O
    8. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
    9. LB medium (Lennox) (Carl Roth, catalog number: X964 )
    10. Ampicillin (Carl Roth, catalog number: K029 )
    11. Kanamycin (Sigma-Aldrich, catalog number: K4000 )
    12. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Carl Roth, catalog number: 2316 )
    13. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957 )
    14. Potassium chloride (KCl)
    15. Sodium phosphate dibasic (Na2HPO4)
    16. Potassium phosphate monobasic (KH2PO4)
    17. LB medium + ampicillin + kanamycin
      1. LB medium (see Recipes)
      2. 100 µg/ml ampicillin (see Recipes)
      3. 25 µg/ml kanamycin (see Recipes)
    18. 0.1 M IPTG (see Recipes)
    19. 1x phosphate-buffered saline (PBS) (see Recipes)

  2. Purification of bacterial protein kinase
    1. Microtubes
      1.5 ml (SARSTEDT, catalog number: 72.690.001 )
      2 ml (SARSTEDT, catalog number: 72.694.006 )
    2. Polypropylene columns 1 ml (QIAGEN, catalog number: 34924 )
    3. Spectra/Por® dialysis membrane MWCO: 3.5-5 kD (Biotech CE Trial Kit) (Spectrum, catalog number: 131201T ), stored at 4 °C in humid atmosphere
    4. Protino Glutathione agarose-4B (Macherey-Nagel, catalog number: 745500.10 ), stored at 4 °C
    5. Glycerol (Carl Roth, catalog number: 3783 )
    6. Tris(hydroxymethyl)aminomethane (Tris) (Carl Roth, catalog number: 5429 )
    7. Glutathione (Sigma-Aldrich, catalog number: G4251 )
    8. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957 )
    9. 1x PBS (see Recipes)
    10. GST elution buffer (see Recipes)
    11. Dialysis buffer (see Recipes)

  3. Purification of ARP2/3 complex
    1. Polypropylene columns 1 ml (QIAGEN, catalog number: 34924 )
    2. PD-10 columns (GE Healthcare, catalog number: 17085101 )
    3. Actin-depleted cellular lysates prepared from Acanthamoeba castellanii (Zuchero, 2007)
    4. Liquid nitrogen (liquid N2)
    5. Tris (Fisher Scientific, catalog number: BP152-5 )
    6. Magnesium chloride (MgCl2) (Fisher Scientific, catalog number: BP214-500 )
    7. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Fisher Scientific, catalog number: BP2939100 )
    8. Dithiothreitol (DTT) (Fisher Scientific, catalog number: BP172-5 )
    9. Glycerol (Fisher Scientific, catalog number: P2294 )
    10. Potassium chloride (KCl) (Fisher Scientific, catalog number: P217-500 )
    11. Adenosine triphosphate (ATP) (Fisher Scientific, catalog number: BP413-25 )
    12. N-WASP VCA-coupled-CH-Sepharose (Zuchero, 2007)
    13. Phenyl Sepharose (Sigma-Aldrich, catalog number: P2459 )
    14. Buffer A (see Recipes)
    15. ARP2/3 elution buffer (see Recipes)
    16. ARP2/3 storage buffer (see Recipes)

  4. Dephosphorylation of ARP2/3 complex
    1. Microtubes (1.5 ml)
    2. 3,500 MWCO Mini dialysis units (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 69550 )
    3. 2 mM Tris, pH 8.0 (Fisher Scientific, catalog number: BP152-5 )
    4. Antarctic phosphatase (New England Biolabs, catalog number: M0289 )
    5. N-WASP VCA-coupled Sepharose (Zuchero, 2007)
    6. Tris (Fisher Scientific, catalog number: BP152-5 )
    7. dH2O
    8. Magnesium chloride hexahydrate (MgCl2) (Fisher Scientific, catalog number: BP214-500 )
    9. Zinc chloride (ZnCl2) (Sigma Aldrich, catalog number: Z3500 )*
    10. 5x HipH buffer (see Recipes)
    11. Buffer A (see Recipes)
    12. ARP2/3 elution buffer (see Recipes)

  5. In vitro phosphorylation assays
    1. Microtubes (1.5 ml) (SARSTEDT, catalog number: 72.690.001 )
    2. Purified bacterial kinase and its catalytic variant
    3. Purified and purified/dephosphorylated ARP2/3 complex
    4. Tris (Carl Roth, catalog number: 5429 )
    5. Manganese chloride (MnCl2) (Fisher Scientific, catalog number: M87-100 )
    6. DTT (Carl Roth, catalog number: 6908 )
    7. ATP (Sigma-Aldrich, catalog number: A5394 )*
    8. dH2O
    9. Magnesium chloride hexahydrate (MgCl2) (Carl Roth, catalog number: 2189 )
    10. Zinc chloride (ZnCl2) (Sigma-Aldrich, catalog number: 211273 )
    11. Sodium dodecyl sulfate (SDS) (Carl Roth, catalog number: 2326 )
    12. Glycerol (Carl Roth, catalog number: 3783 )
    13. Bromophenol blue (Sigma-Aldrich, catalog number: B6131 )*
    14. β-mercaptoethanol (Sigma-Aldrich, catalog number: O34461-100 )
    15. Antarctic phosphatase (New England Biolabs, catalog number: M0289 )
    16. 10x phosphorylation buffer (see Recipes)
    17. ATP solution at 0.5 mg.ml-1 (see Recipes)
    18. 5x Laemmli loading buffer (see Recipes)
    19. 5x HipH buffer (see Recipes)

  6. Detection of phosphorylation level with Western Blot
    1. Square Petri dish (Greiner Bio One International, catalog number: 688162 )
    2. Whatman 3 MM CHR paper (GE Healthcare, catalog number: 3030-917 )
    3. Nitrocellulose membrane Optitran BA-S85 reinforced NC (GE Healthcare, catalog number: 10439194 )
    4. 50 ml conical tubes
    5. Parafilm
    6. Plastic wrap
    7. Page RulerTM prestained protein ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26616 )
    8. Monoclonal mouse anti-phosphothreonine antibody, Clone PTR-8 (Sigma-Aldrich, catalog number: P6623 )
    9. Goat anti-mouse IgG peroxidase antibody (Sigma-Aldrich, catalog number: A0168 )
    10. Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 34080 )
    11. Rotiphorese Gel 30 (30% acrylamide, 0.8% bisacrylamide) (Carl Roth, catalog number: 3029 )
    12. Tris (Carl Roth, catalog number: 5429 )
    13. dH2O
    14. SDS (Carl Roth, catalog number: 2326 )
    15. Ammonium persulfate (APS) (Carl Roth, catalog number: 9592 )
    16. Tetramethylethylenediamine (TEMED) (Carl Roth, catalog number: 2367 )
    17. Glycine (Carl Roth, catalog number: 0079 )
    18. Glacial acetic acid
    19. Ethanol
    20. Coomassie Brilliant Blue R-250 (MP Biomedicals, catalog number: 1-800-854-0530 or MP Biomedicals, catalog number: 02190682 )
    21. Methanol
    22. 6-aminohexanoic acid
    23. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957 )
    24. Bovine serum albumin (BSA) (Carl Roth, catalog number: T844 )
    25. Tween 20 (VWR, BDH®, catalog number: 663684B )
    26. Anti-phosphotyrosine antibody (EMD Millipore, catalog number: AB1607 )
    27. Migration buffer (see Recipes)
    28. 12% SDS-PAGE gels (see Recipes)
    29. Staining solution (see Recipes)
    30. Destaining solution (see Recipes)
    31. Assembly for the semi-dry transfer
      a. Transfer solution 1 (see Recipes)
      b. Transfer solution 2 (see Recipes)
      c. Transfer solution 3 (see Recipes)
    32. Tris-buffered saline (TBS) (see Recipes)
    33. TBS-5% BSA (see Recipes)
    34. TBS-0.1% Tween 20 (see Recipes)
    35. Anti-phosphothreonine antibodies solution (see Recipes)
    36. Anti-mouse-peroxydase antibodies solution (see Recipes)

*Note: These products have been discontinued.

Equipment

  1. Shaker/incubator at 37 °C and 20 °C for tubes and 500 ml Erlenmeyer flask
  2. Autoclave
  3. Ultrospec 10-cell density meter (GE Healthcare, Amersham Biosciences, model: Ultrospec® 10 )
  4. Centrifuges for conical and microtubes at 4 °C
  5. French pressure cell press (American instrument company)
  6. Tubes rotator for conical and microtubes
  7. Support of purification columns
  8. NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
  9. Freezer -80 °C
  10. Lab water bath at 37 °C and 30 °C
  11. Dry block heater at 100 °C
  12. HoeferTM Dual Gel Caster system (GE Healthcare, Amersham Biosciences, model: Dual Gel Caster )
  13. Electrophoresis power supply
  14. Semi-dry blotter (C.B.S Scientific, catalog number: EBU-4000 ) with GD3000 D generator (Sebia)
  15. ChemiStart 5000 (Fisher Bioblock Scientific)

Procedure

  1. Production and extraction of bacterial protein kinase
    To test the ability of a protein kinase to phosphorylate ARP2/3 complex, the protein must be overexpressed in the appropriate cell expression system and purified. For example, the gene encoding a protein kinase may be cloned in pGEX-6P-3 plasmid to produce a fusion protein with an N-terminal GST tag (the BamHI/SalI restriction sites can be often used in our cases). A catalytic variant of the protein kinase, with amino acid substitutions that abolish the kinase activity, should be generated from the same construct and used as a control. Mutagenesis may be accomplished using the QuikChange II site-directed mutagenesis kit (Stratagene), per the manufacturer’s instructions, to substitute the invariant lysine essential for donor-ATP binding with a methionine. The genetic constructs are then transformed in BL21 (pREP4-groESL) E.coli strain, which expresses the GroES and GroEL chaperon proteins to promote folding of the overproduced fusion protein of interest, here the GST-protein kinase.
    Note: The pGEX-6P-3 plasmid is not always adaptable for the cloning and expression of your protein. In such cases, the pQE30 plasmid can also be used to generate a 6His affinity-tagged protein kinase. The purification protocol should then be adapted to include purification by immobilized metal affinity media (i.e., nickel beads).
    1. Inoculate 5 ml of LB medium containing 100 µg/ml of ampicillin and 25 µg/ml of kanamycin with one colony of the overproducing strain described above.
    2. Incubate the culture overnight at 37 °C with agitation.
    3. Inoculate 100 ml of LB + ampicillin + kanamycin with 2 ml of overnight bacterial culture (dilution 1/50).
    4. Incubate the culture at 37 °C with agitation until the optical density reaches an OD600 nm of 0.7.
    5. Add 1 ml of 0.1 M IPTG (1 mM final concentration) in the culture to induce the overproduction of recombinant protein.
    6. Incubate for additional 4 h at 20 °C with agitation.
    7. Split the culture content equally in two conical tubes of 50 ml.

    Note: For all remaining steps, the samples should be kept on in ice.

    1. Centrifuge the two tubes at 6,000 x g for 10 min at 4 °C.
    2. Decant the supernatants.
    3. Wash each bacteria pellet with 10 ml of prechilled dH2O.
    4. Pool the two pellets in one conical tube.
    5. Centrifuge at 6,000 x g for 10 min at 4 °C.
    6. Decant the supernatant.
    7. Resuspend the bacteria with 5 ml of prechilled 1x PBS and 50 µl of protease inhibitor cocktail.
    8. Lyse the bacteria by two passages through a French press.
    9. Centrifuge the extract for 30 min at 14,000 x g at 4 °C.
    10. Collect the supernatant that contains the soluble overproduced proteins.

  2. Purification of bacterial protein kinase
    1. Equilibrate the glutathione agarose-4B resin:
      1. Pipette 250 µl resin in a microtube for each protein kinase to purify.
      2. Centrifuge 5 min at 1,000 x g.
      3. Discard the supernatants.
      4. Add 1 ml of 1x PBS.
      5. Gently invert the resins until well resuspended.
      6. Centrifuge 5 min at 1,000 x g.
      7. Repeat washing with 1x PBS 3 times.
    2. Load the supernatant containing overproduced proteins on the equilibrated glutathione resin.
    3. Incubate the proteins/resin mix for 4 h at 4 °C with gentle rotation.
    4. Load the proteins/resin mix on a column and allow the column to empty by gravity flow to separate the resin from the flow-through.
    5. Collect the flow-through and load into the column a second time to collect as much recombinant protein as possible.
    6. Wash the resin with 5 ml of 1x PBS. Allow PBS to move through the column by gravity flow. Repeat this washing step 3 times.
    7. Elute the GST tagged protein kinase with 1 ml of GST elution buffer twice and keep the sample in ice.
    8. Dialyze the eluate using a dialysis membrane:
      1. Cut the appropriate length of dialysis membrane.
      2. Incubate the membrane for 1 h in dH2O with gentle agitation to rinse.
      3. Place the eluate in a microtube, puncture a large hole in the cap and replace with the dialysis membrane.
      4. Invert the microtube (with dialysis membrane towards the buffer) in 1,000 volumes of dialysis buffer and dialyze overnight at 4 °C.
    9. Determine the protein concentration with a NanoDrop assay.
      Note: Any method to determine the protein concentration may be used.
    10. Dispense 100 µl aliquots of the protein into 1.5 ml prechilled microfuge tubes.
    11. Add 25 µl of glycerol (20% final).
    12. Store the proteins at -80 °C until needed.


      Figure 1. Example of purified bacterial protein kinase. Coomassie staining of SDS-PAGE gel with an overexpressed and purified bacterial protein kinase from the Legionella pneumophila bacterium, GST-LegK2. MW: molecular weight, NI: non-induced fraction of bacteria, I: induced fraction of bacteria, E: eluted fraction.

  3. Purification of ARP2/3 complex
    1. All reagents and the purification of ARP2/3 complex must be performed at 4 °C.
    2. Equilibrate a 1 ml polypropylene column containing VCA-coupled Sepharose with buffer A.
    3. Apply actin-depleted extracts of Acanthamoeba castellanii to the column at 4 °C.
      Note: Actin-depleted extracts of Acanthamoeba castellanii prepared as previously described in Zuchero (2007). In vitro actin assembly assays and purification from Acanthamoeba. Methods Mol Biol 370: 213-226.
    4. Wash with 20 column volumes of buffer A.
    5. Elute ARP2/3 with ARP2/3 elution buffer.
    6. Pass ARP2/3 enriched fractions over a 1 ml phenyl Sepharose column equilibrated in elution buffer followed by 1.5 column volumes of elution buffer.
    7. Collect the filtrate and identify fractions enriched in ARP2/3 by SDS-PAGE.
    8. Exchange ARP2/3 complex enriched fractions into ARP2/3 storage buffer using a PD10 column or dialysis.
    9. Aliquot ARP2/3 complex, flash freeze in liquid N2 and store at -80 °C.


      Figure 2. Purified ARP2/3 complex. Coomassie staining of SDS-PAGE gel with ARP2/3 complex purified from Acanthamoeba castellanii. MW: molecular weight.

  4. Dephosphorylation of ARP2/3 complex
    1. Dilute ARP2/3 complex 1:1 with 2 mM Tris pH 8.0 and combine with HipH buffer containing 1 U Antarctic phosphatase.
    2. Incubate at 30 °C for 1.5 h.
    3. Mock-treated controls should be incubated with heat-inactivated phosphatase at 65 °C for 30 min.
    4. Affinity purify dephosphorylated ARP2/3 complex using N-WASP VCA-coupled to activated CH-Sepharose.
    5. Elute ARP2/3 complex from Sepharose with ARP2/3 elution buffer.
    6. Dialyze ARP2/3 against buffer A overnight at 4 °C.

  5. In vitro phosphorylation assays
    1. Prepare the following reactions in duplicate in 1.5 ml microtubes:

      Table 1. Reactions for the phosphorylation assays


    2. Incubate the reactions for 30 min at 37 °C in water bath.
    3. In the phosphatase test tubes, add 2 µl of Antarctic phosphatase at 5,000 U/ml (10 U final) with 5 µl of its 5x HipH buffer and incubate for an additional 1 h at 30 °C in water bath.
    4. Stop the reactions by the addition of 5 µl of 5x Laemmli loading buffer.

  6. Detection of phosphorylation level by Western blot with an anti-phosphothreonine antibody
    1. Prepare two SDS-PAGE gels at 12%.
    2. Heat the samples for 5 min at 100 °C in dry block heater.
    3. Load 20 µl of heated samples and 5 µl of protein ladder in gel and electrophoresis in SDS-PAGE migration buffer at 35 mA by gel for approximately 45 min.
      Note: The amperage and time of migration are dependent of the gel size.
    4. Stain one of the gels with Coomassie blue in a Petri dish.
      1. Stain the gel for 20 min in staining solution with gentle agitation.
      2. Wash the gel for 30 min to 1 h with gentle agitation in destaining solution.
      3. Analyze the different fractions.
    5. Perform a Western blot with the second gel.
      1. Transfer the proteins from the second gel to a nitrocellulose membrane in semi-dry conditions with 0.8 mA/cm2 for 1 h.
      2. Place the membrane in a dish adapted for agitation or a conical tube. The side of membrane with proteins must not be in contact with the container and the solutions must completely cover the membrane.
      3. Block the membrane for 1 h at room temperature with gentle rotation in TBS-5% BSA.
      4. Wash the membrane 3 x 5 min in TBS-0.1% Tween 20.
      5. Incubate the membrane for 1 h at room temperature with gentle rotation in anti-phosphothreonine antibodies solution.
        Note: Following this protocol, we detect threonine phosphorylation level. Tyrosine phosphorylation level of ARP2/3 complex can also be detected using an anti-phosphotyrosine antibody (EMD Millipore, catalog number: AB1607 ) or another anti-phosphotyrosine antibody, adjust the procedure to follow the manufacturer’s recommendations if necessary.
      6. Wash the membrane 3 x 5 min in TBS-0.1% Tween 20.
      7. Incubate the membrane for 1 h at room temperature with gentle rotation in anti-mouse-peroxidase antibody solution.
      8. Wash the membrane 3 x 5 min in TBS-0.1% Tween 20.
      9. Wash the membrane with TBS.
      10. Detect the anti-phosphothreonine labeling with SuperSignal West Pico Chemiluminescent Substrate Kit.
        1. Place 1 ml of chemiluminescent substrate (500 µl of stable peroxide solution + 500 µl of Luminol/Enhancer solution) on a sheet of Parafilm.
        2. Place the membrane on the chemiluminescent substrate directly so that the proteins are in direct contact with the peroxidase substrate.
          Note: Caution, remove any bubbles that are present between the membrane and the substrate.
        3. Incubate 5 min in the dark.
        4. Drain the solution from the membrane and place it on a support.
        5. Cover the membrane with plastic wrap to prevent drying.
        6. Image membrane at different exposures with a ChemiStart or appropriate detection system.
        Notes:
        1. Other methods for detecting the chemiluminescent signal may be used according to recommendations by the equipment manufacturer.
        2. You may also detect the autophosphorylation state of your protein kinase. However, we suggest that the analysis be performed at a different polyacrylamide concentration if the molecular weights of the kinase and its substrates are substantially different.
        3. The original publication describing ARP2/3 phosphorylation can also be help to set the conditions and for the analysis: LeClaire, L. L. 3rd, Baumgartner, M., Iwasa, J. H., Mullins, R. D., Barber, D. L. (2008). Phosphorylation of the Arp2/3 complex is necessary to nucleate actin filaments. J Cell Biol 182(4):647-54.

Notes

  1. To optimize the expression and purification of bacterial protein, all the fractions corresponding to each steps of protocol may be analyzed by Coomassie blue staining after SDS-PAGE.
  2. The bacterial pellet of step A13 can be frozen at -80 °C.
  3. For our ARP2/3 phosphorylation assays, we used the anti-phosphothreonine antibody from Sigma. However, we found that labeling by the antibody is inconsistent and requires optimization of the experimental conditions. To test the phosphorylation activity, we found that the polyclonal phosphothreonine antibody from Cell Signaling (#9381) showed more consistent labeling patterns and was more specific for labeling.
  4. Several steps of the Western blot analysis can be performed overnight; the transfer (at 0.2 mA/cm2); blocking (at 4 °C); or incubation with the primary antibody (at 4 °C).
  5. Wear gloves to protect the samples from proteases and/or contaminating proteins.

Recipes

  1. LB medium + ampicillin (100 µg/ml) + kanamycin (25 µg/ml)
    0.2 ml of 100 mg/ml ampicillin stock solution
    25 µl of 50 mg/ml kanamycin stock solution
    200 ml of LB medium
    1. LB medium
      20 g of LB medium (Lennox)
      1 L of dH2O
      Aliquot by 200 ml
      Sterilized by autoclaving
    2. 100 mg/ml ampicillin stock solution
      5 g of ampicillin
      50 ml of dH2O
      Sterilize by filtration
      Aliquot and freeze the solution until needed
    3. 50 mg/ml kanamycin stock solution
      2.5 g of kanamycin
      50 ml of dH2O
      Sterilize by filtration
      Aliquot and freeze the solution until needed
  2. 0.1 M IPTG
    238 mg of IPTG
    10 ml of dH2O
    Sterilize by filtration
    Aliquot and freeze until needed
  3. 10x PBS (1 L)
    80 g NaCl
    2 g KCl
    14.4 g Na2HPO4
    2.4 g KH2PO4
    Adjust pH to 7.4
    Add dH2O to 1 L
  4. 1x PBS (1 L)
    100 ml of 10x PBS
    900 ml of dH2O
  5. GST elution buffer
    50 mM Tris HCl pH 8.0
    10 mM glutathione
  6. Dialysis buffer
    50 mM Tris HCl pH 7.5
    150 mM NaCl
    10 % glycerol
  7. ARP2/3 elution buffer
    10 mM Tris, pH 8.0
    400 mM MgCl2
  8. ARP2/3 storage buffer
    1 mM HEPES
    1 mM DTT
    10% glycerol
    Adjust pH to 7.0
  9. 10x phosphorylation buffer
    250 mM Tris HCl pH 7.5
    50 mM MnCl2
    50 mM DTT
  10. ATP solution at 0.5 mg/ml
    1 mg of ATP
    2 ml of dH2O
    Prepare immediately before use
  11. 5x HipH buffer
    50 mM Tris pH 8.0
    1 mM Mg2Cl2
    0.1 mM ZnCl2
  12. Buffer A 10x solution
    500 mM KCl
    20 mM MgCl2
    10 mM ATP
    100 mM Tris, pH 7.5
  13. 5x Laemmli loading buffer
    5 ml 10% SDS
    6.25 ml 0.5 M Tris HCl pH 6.8
    5 ml glycerol
    7.5 ml dH2O
    1 pinch of bromophenol blue
    2.5 µl 1 M β-mercaptoethanol
  14. 12% SDS-PAGE gels (for 2 mini-gels)
    1. Stacking gels
      1 ml 30% ProtoGel (30% acrylamide, 0.8% bisacrylamide)
      1.25 ml 0.5 M Tris HCl pH 6.8
      2.65 ml dH2O
      50 µl 10% SDS
      50 µl 10% APS
      5 µl TEMED
    2. Separating gels
      4 ml 30% ProtoGel (30% acrylamide, 0.8% bisacrylamide)
      2.5 ml 1.5 M Tris HCl pH 8.8
      3 ml dH2O
      100 µl 10% SDS
      100 µl 10% APS
      10 µl TEMED
  15. Migration buffer
    25 mM Tris
    192 mM glycine
    0.1% SDS
  16. Staining solution
    10% glacial acetic acid
    40% ethanol
    0.04% Coomassie Brilliant Blue R-250
  17. Destaining solution
    10% glacial acetic acid
    5% ethanol
  18. Transfer solution 1
    300 mM Tris
    20% methanol
  19. Transfer solution 2
    25 mM Tris
    20% methanol
  20. Transfer solution 3
    25 mM Tris
    40 mM 6-aminohexanoic acid
    20% methanol
  21. Assembly for the semi-dry transfer
    Cathode (-) 

    Anode (+)
  22. TBS
    100 mM Tris
    150 mM NaCl
    Adjust pH to 8
  23. TBS-5% BSA
    0.5 g of BSA
    10 ml TBS
  24. TBS-0.1% Tween 20
    1 ml of Tween 20
    1 L TBS
  25. Anti-phosphothreonine antibodies solution
    PBS-5% BSA-0.1% Tween 20
    Monoclonal mouse anti-phosphothreonine antibody (1/500)
  26. Anti-mouse-peroxydase antibodies solution
    TBS-5% BSA-0.1% Tween 20
    Goat anti-mouse IgG peroxydase antibody (1/5,000)

Acknowledgments

This work was performed within the framework of the LABEX ECOFECT (ANR-11-LABX-0042) of the Université de Lyon, within the program Investissements d’avenir (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). This work was funded by the Centre National de la Recherche Scientifique (UMR 5308), the Institut National de la Recherche Médicale (U1111) and the Université Lyon 1.
This protocol was derived from previously described protocols: (Hervet et al., 2011; Zuchero, 2007 and LeClaire et al., 2008)

References

  1. Amrein, K. E., Takacs, B., Stieger, M., Molnos, J., Flint, N. A. and Burn, P. (1995). Purification and characterization of recombinant human p50csk protein-tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL. Proc Natl Acad Sci U S A 92(4): 1048-1052.
  2. Haglund, C. M. and Welch, M. D. (2011). Pathogens and polymers: microbe-host interactions illuminate the cytoskeleton. J Cell Biol 195(1): 7-17.
  3. Hervet, E., Charpentier, X., Vianney, A., Lazzaroni, J. C., Gilbert, C., Atlan, D. and Doublet, P. (2011). Protein kinase LegK2 is a type IV secretion system effector involved in endoplasmic reticulum recruitment and intracellular replication of Legionella pneumophila. Infect Immun 79(5): 1936-50.
  4. LeClaire, L. L., 3rd, Baumgartner, M., Iwasa, J. H., Mullins, R. D. and Barber, D. L. (2008). Phosphorylation of the Arp2/3 complex is necessary to nucleate actin filaments. J Cell Biol 182(4): 647-654.
  5. LeClaire, L. L., Rana, M., Baumgartner, M. and Barber, D. L. (2015). The Nck-interacting kinase NIK increases Arp2/3 complex activity by phosphorylating the Arp2 subunit. J Cell Biol 208(2): 161-170.
  6. Michard, C., Sperandio, D., Bailo, N., Pizarro-Cerda, J., LeClaire, L., Chadeau-Argaud, E., Pombo-Gregoire, I., Hervet, E., Vianney, A., Gilbert, C., Faure, M., Cossart, P. and Doublet, P. (2015). The legionella kinase LegK2 targets the ARP2/3 complex to inhibit actin nucleation on phagosomes and allow bacterial evasion of the late endocytic pathway. MBio 6(3): e00354-00315.
  7. Narayanan, A., LeClaire, L. L., 3rd, Barber, D. L. and Jacobson, M. P. (2011). Phosphorylation of the Arp2 subunit relieves auto-inhibitory interactions for Arp2/3 complex activation. PLoS Comput Biol 7(11): e1002226.
  8. Smith, D. B. and Johnson, K. S. (1988). Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67(1): 31-40.
  9. Vadlamudi, R. K., Li, F., Barnes, C. J., Bagheri-Yarmand, R. and Kumar, R. (2004). p41-Arc subunit of human Arp2/3 complex is a p21-activated kinase-1-interacting substrate. EMBO Rep 5(2): 154-160.
  10. Welch, M. D. and Way, M. (2013). Arp2/3-mediated actin-based motility: a tail of pathogen abuse. Cell Host Microbe 14(3): 242-255.
  11. Zuchero, J. B. (2007). In vitro actin assembly assays and purification from Acanthamoeba. Methods Mol Biol 370: 213-226.

简介

肌动蛋白相关蛋白2/3(ARP2 / 3)复合物是在哺乳动物细胞中产生支链肌动蛋白网络的肌动蛋白成核剂。除了结合成核促进因子之外,LeClaire等人。证明其磷酸化状态是其活性的关键(LeClaire等人,2008)。在细胞中,ARP2 / 3复合物在ARP2,ARP3和ARPC1亚基的苏氨酸和酪氨酸残基上磷酸化(Vadlamudi等人,2004; LeClaire等人)。 ,2008; Narayanan等人,2011; LeClaire等人,2015)。特别地,ARP2亚基的苏氨酸237和238的磷酸化对于允许将ARP2 / 3复合物结构改变为其活性构象是必要的(Narayanan等人,2011; LeClaire等人al ,2015)。虽然对于真核细胞中的许多功能很重要,但ARP2 / 3复合物活性也有利于多种细胞病原体(Haglund和Welch,2011; Welch和Way,2013)。最近,我们证明细菌病原体,嗜肺军团菌,使用注射在宿主细胞质细胞中的细菌蛋白激酶来操纵ARP2 / 3复合磷酸化状态(Michard等人,2015) )。在这里,我们描述如何测试细菌蛋白激酶或另一种蛋白激酶在体外上下文中磷酸化ARP2 / 3复合物的能力。首先,产生和纯化ARP2 / 3复合物和细菌蛋白激酶。然后,将纯化的蛋白质在ATP存在下培养,并通过Western印迹分析ARP2 / 3磷酸化水平。

ARP2 / 3复合物在苏氨酸和酪氨酸残基上磷酸化(LeClaire等人,2008)。 ARP2 / 3复合物的ARPC1和ARP2亚基上的四个磷酸化位点目前已知:ARPC1的苏氨酸21,ARP2的苏氨酸237/238和酪氨酸202各自表现出对ARP2 / 3功能的重要作用(Vadlamudi et al。 ,2004; LeClaire等人,2008; Narayanan等人,2011)。我们实验室最近的一项研究表明,军团菌激酶2(LegK2)是嗜肺军团杆菌的效应物丝氨酸/苏氨酸蛋白激酶修饰ARPC1B和ARP3的苏氨酸磷酸化状态ARP2 / 3复合体亚单位。 ARP2 / 3复合磷酸化使细胞空泡失活并阻断肌动蛋白聚合,防止细胞内吞途径的降解(Michard等人,2015)。该协议描述了在我们的实验室中常规使用的体外磷酸化测定法,以测试作为ARP2 / 3复合物亚基的蛋白激酶的潜在底物。该方案还可以根据需要调整其他底物。

关键字:体外磷酸化测定, 蛋白激酶, ARP2/3复合物, 蛋白质纯化, 蛋白质印迹分析

材料和试剂

  1. 过量生产和提取细菌蛋白激酶
    1. 50ml锥形管(Greiner Bio One International,目录号:227261)
    2. 无菌培养管(SARSTEDT,目录号:62.515.006)
    3. 灭菌过滤器(0.22μm)(Dutscher Scientific,目录号:51732)
    4. 分光光度计比色皿(Dutscher Scientific,目录号:030101)
    5. 大肠杆菌BL21(pREP4-groESL)(Amrein等人,1995)
    6. pGEX-6P-3(Smith和Johnson,1988)/pGEX-蛋白激酶
    7. 预冷dH 2 O
    8. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
    9. LB培养基(Lennox)(Carl Roth,目录号:X964)
    10. 氨苄青霉素(Carl Roth,目录号:K029)
    11. 卡那霉素(Sigma-Aldrich,目录号:K4000)
    12. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Carl Roth,目录号:2316)
    13. 氯化钠(NaCl)(Carl Roth,目录号:3957)
    14. 氯化钾(KCl)
    15. 磷酸氢二钠(Na 2 HPO 4)
    16. 磷酸二氢钾(KH 2 PO 4)
    17. LB培养基+氨苄西林+卡那霉素
      1. LB培养基(参见食谱)
      2. 100μg/ml氨苄青霉素(参见食谱)
      3. 25μg/ml卡那霉素(见配方)
    18. 0.1 M IPTG(见配方)
    19. 1x磷酸盐缓冲盐水(PBS)(见食谱)

  2. 细菌蛋白激酶的纯化
    1. 微管
      1.5毫升(SARSTEDT,目录号:72.690.001)
      2毫升(SARSTEDT,目录号:72.694.006)
    2. 聚丙烯柱1ml(QIAGEN,目录号:34924)
    3. Spectra/Por 透析膜MWCO:3.5-5kD(Biotech CE试剂盒)(Spectrum,目录号:131201T),在4℃在潮湿气氛中储存
    4. Protino谷胱甘肽琼脂糖-4B(Macherey-Nagel,目录号:745500.10),储存在4℃下
    5. 甘油(Carl Roth,目录号:3783)
    6. 三羟甲基氨基甲烷(Tris)(Carl Roth,目录号:5429)
    7. 谷胱甘肽(Sigma-Aldrich,目录号:G4251)
    8. 氯化钠(NaCl)(Carl Roth,目录号:3957)
    9. 1x PBS(见食谱)
    10. GST洗脱缓冲液(参见食谱)
    11. 透析缓冲液(见配方)

  3. 净化ARP2/3复合体
    1. 聚丙烯柱1ml(QIAGEN,目录号:34924)
    2. PD-10色谱柱(GE Healthcare,目录号:17085101)
    3. 由Acanthamoeba castellanii制备的肌动蛋白耗尽的细胞裂解物(Zuchero,2007)
    4. 液氮(液氮N 2)
    5. Tris(Fisher Scientific,目录号:BP152-5)
    6. 氯化镁(MgCl 2)(Fisher Scientific,目录号:BP214-500)
    7. 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)(Fisher Scientific,目录号:BP2939100)
    8. 二硫苏糖醇(DTT)(Fisher Scientific,目录号:BP172-5)
    9. 甘油(Fisher Scientific,目录号:P2294)
    10. 氯化钾(KCl)(Fisher Scientific,目录号:P217-500)
    11. 三磷酸腺苷(ATP)(Fisher Scientific,目录号:BP413-25)
    12. N-WASP VCA偶联CH-Sepharose(Zuchero,2007)
    13. 苯基琼脂糖凝胶(Sigma-Aldrich,目录号:P2459)
    14. 缓冲液A(参见食谱)
    15. ARP2/3洗脱缓冲液(参见食谱)
    16. ARP2/3存储缓冲区(见配方)

  4. ARP2/3复合物的脱磷酸酯
    1. 微管(1.5 ml)
    2. 3,500 MWCO迷你透析装置(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:69550)
    3. 2mM Tris,pH 8.0(Fisher Scientific,目录号:BP152-5)
    4. 南极磷酸酶(New England Biolabs,目录号:M0289)
    5. N-WASP VCA偶联Sepharose(Zuchero,2007)
    6. Tris(Fisher Scientific,目录号:BP152-5)
    7. dH 2 O
    8. 氯化镁六水合物(MgCl 2)(Fisher Scientific,目录号:BP214-500)
    9. 氯化锌(ZnCl 2)(Sigma Aldrich,目录号:Z3500)*
    10. 5x HipH缓冲液(见配方)
    11. 缓冲液A(参见食谱)
    12. ARP2/3洗脱缓冲液(参见食谱)

  5. 磷酸化测定
    1. 微管(1.5ml)(SARSTEDT,目录号:72.690.001)
    2. 纯化细菌激酶及其催化变体
    3. 纯化和纯化/脱磷酸化的ARP2/3复合物
    4. Tris(Carl Roth,目录号:5429)
    5. 氯化锰(MnCl 2)(Fisher Scientific,目录号:M87-100)
    6. DTT(Carl Roth,目录号:6908)
    7. ATP(Sigma-Aldrich,目录号:A5394)*
    8. dH 2 O
    9. 氯化镁六水合物(MgCl 2)(Carl Roth,目录号:2189)
    10. 氯化锌(ZnCl 2)(Sigma-Aldrich,目录号:211273)
    11. 十二烷基硫酸钠(SDS)(Carl Roth,目录号:2326)
    12. 甘油(Carl Roth,目录号:3783)
    13. 溴苯酚蓝(Sigma-Aldrich,目录号:B6131)*
    14. β-巯基乙醇(Sigma-Aldrich,目录号:O34461-100)
    15. 南极磷酸酶(New England Biolabs,目录号:M0289)
    16. 10x磷酸化缓冲液(参见食谱)
    17. ATP溶液为0.5mg.ml -1 (参见食谱)
    18. 5x Laemmli加载缓冲区(请参阅配方)
    19. 5x HipH缓冲液(见配方)

  6. 用Western Blot检测磷酸化水平
    1. 方格培养皿(Greiner Bio One International,目录号:688162)
    2. Whatman 3 MM CHR纸(GE Healthcare,目录号:3030-917)
    3. 硝酸纤维素膜Optitran BA-S85增强型NC(GE Healthcare,目录号:10439194)
    4. 50ml圆锥管
    5. 石蜡膜
    6. 塑料包装
    7. 页面标尺 TM 预饱和蛋白梯(Thermo Fisher Scientific,Thermo Scientific TM,目录号:26616)
    8. 单克隆小鼠抗磷酸苏氨酸抗体,克隆PTR-8(Sigma-Aldrich,目录号:P6623)
    9. 山羊抗小鼠IgG过氧化物酶抗体(Sigma-Aldrich,目录号:A0168)
    10. 超信号西微微化学发光底物(Thermo Fisher Scientific,Thermo Scientific TM,目录号:34080)
    11. Rotiphorese凝胶30(30%丙烯酰胺,0.8%双丙烯酰胺)(Carl Roth,目录号:3029)
    12. Tris(Carl Roth,目录号:5429)
    13. dH 2 O
    14. SDS(Carl Roth,目录号:2326)
    15. 过硫酸铵(APS)(Carl Roth,目录号:9592)
    16. 四甲基乙二胺(TEMED)(Carl Roth,目录号:2367)
    17. 甘氨酸(Carl Roth,目录号:0079)
    18. 冰醋酸
    19. 乙醇
    20. 考马斯亮蓝R-250(MP Biomedicals,目录号:1-800-854-0530或MP Biomedicals,目录号:02190682)
    21. 甲醇
    22. 6-氨基己酸
    23. 氯化钠(NaCl)(Carl Roth,目录号:3957)
    24. 牛血清白蛋白(BSA)(Carl Roth,目录号:T844)
    25. 吐温20(VWR,BDH ®,目录号:663684B)
    26. 抗磷酸酪氨酸抗体(EMD Millipore,目录号:AB1607)
    27. 迁移缓冲区(见配方)
    28. 12%SDS-PAGE凝胶(见配方)
    29. 染色溶液(参见食谱)
    30. 解决方案(见配方)
    31. 组装半干运输
      一个。转移解决方案1(见配方)
      b。转移解决方案2(见配方)
      C。转移解决方案3(见配方)
    32. Tris缓冲盐水(TBS)(见食谱)
    33. TBS-5%BSA(参见食谱)
    34. TBS-0.1%吐温20(参见食谱)
    35. 抗磷酸核糖抗体溶液(参见食谱)
    36. 抗小鼠过氧化物酶抗体溶液(参见食谱)

注意:这些产品已经停产。

设备

  1. 振荡器/培养箱在37℃和20℃用于管和500ml锥形瓶
  2. 高压灭菌器
  3. Ultrospec 10细胞密度计(GE Healthcare,Amersham Biosciences,型号:Ultrospec 10)
  4. 圆锥和微管在4°C的离心机
  5. 法国压力机(美国仪器公司)
  6. 用于锥形和微管的管旋转器
  7. 支持净化柱
  8. NanoDrop 2000分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000)
  9. 冰箱-80°C
  10. 实验室水浴37°C和30°C
  11. 100°C干块加热器
  12. Hoefer TM双重凝胶脚轮系统(GE Healthcare,Amersham Biosciences,型号:Dual Gel Caster)
  13. 电泳电源
  14. GD3000 D发生器(Sebia)的半干式吸墨纸(C.B.S Scientific,目录号:EBU-4000)
  15. ChemiStart 5000(Fisher Bioblock Scientific)

程序

  1. 细菌蛋白激酶的生产和提取
    为了测试蛋白激酶磷酸化ARP2/3复合物的能力,蛋白质必须在适当的细胞表达系统中过表达并纯化。例如,编码蛋白激酶的基因可以克隆到pGEX-6P-3质粒中以产生具有N-末端GST标签的融合蛋白(在我们的情况下可以经常使用BamHI/SalI限制性位点)。蛋白激酶的催化变体,具有消除激酶活性的氨基酸取代,应该由相同的构建体产生并用作对照。诱变可以使用QuikChange II定点诱变试剂盒(Stratagene)根据制造商的说明书来替代与甲硫氨酸进行供体-ATP结合所必需的不变赖氨酸。然后将遗传构建体转化到表达GroES和GroEL伴侣蛋白的BL21(pREP4-groESL)大肠杆菌菌株中,以促进过量产生的感兴趣的融合蛋白的折叠,这里是GST蛋白激酶 注意:pGEX-6P-3质粒不总是适用于您的蛋白质的克隆和表达。在这种情况下,pQE30质粒也可用于产生6His亲和标记的蛋白激酶。然后,纯化方案应适于包括通过固定的金属亲和介质(即镍珠)的纯化。
    1. 接种含有100μg/ml氨苄青霉素和25μg/ml卡那霉素的5ml含有上述过量产生菌株的LB培养基。
    2. 在37℃下搅拌孵育培养一夜。
    3. 接种100ml的LB +氨苄青霉素+卡那霉素与2ml过夜的细菌培养物(稀释1/50)。
    4. 在37℃下搅拌孵育培养物直到光密度达到0.7的OD 600nm 。
    5. 在培养物中加入1ml 0.1M IPTG(终浓度为1mM)以诱导重组蛋白质的过量产生。
    6. 在20°C下再搅拌孵育另外4小时。
    7. 将培养物质均匀分成两个50ml的锥形管。

    注意:对于所有剩余的步骤,样品应保存在冰上。

    1. 将6,000 x g的两个管在4℃离心10分钟。
    2. 倾倒上清液。
    3. 用10ml预冷的dH 2 O洗涤每个细菌颗粒。
    4. 将两个丸粒放入一个锥形管中。
    5. 在4℃下以6,000×g离心10分钟。
    6. 倾倒上清液。
    7. 用5ml预先预先注入的1x PBS和50μl蛋白酶抑制剂混合物重悬细菌。
    8. 通过法国新闻界两次传播细菌。
    9. 在4℃下以14,000xg离心提取物30分钟。
    10. 收集含有可溶性过量产生蛋白质的上清液
  2. 细菌蛋白激酶的纯化
    1. 平衡谷胱甘肽琼脂糖-4B树脂:
      1. 在每个蛋白激酶的微管中吸取250μl树脂以进行纯化
      2. 离心5分钟,在1,000 x g
      3. 弃上清液。
      4. 加入1毫升1x PBS。
      5. 轻轻倒转树脂,直到重新悬浮。
      6. 以1,000 x g离心5分钟。
      7. 用1x PBS重复洗涤3次。
    2. 将含有过量产生的蛋白质的上清液加载到平衡的谷胱甘肽树脂上。
    3. 蛋白质/树脂混合物在4℃下温和旋转孵育4小时
    4. 将蛋白质/树脂混合物装入柱子上,并通过重力流使色谱柱排空,以将树脂从流通中分离出来。
    5. 收集流过并负载到柱中第二次收集尽可能多的重组蛋白。
    6. 用5ml PBS冲洗树脂。允许PBS通过重力流动通过色谱柱。重复这个洗涤步骤3次。
    7. 用1ml GST洗脱缓冲液洗脱GST标记的蛋白激酶两次,并将样品保持在冰中
    8. 透析膜透析洗脱液:
      1. 切割适当长度的透析膜。
      2. 在轻轻搅拌下将膜在dH 2 O中孵育1小时以冲洗。
      3. 将洗脱液放在微管中,在盖子上穿一个大孔,并用透析膜代替。
      4. 将微管(透析膜朝向缓冲液)倒入1000体积的透析缓冲液中,并在4℃透析过夜。
    9. 用NanoDrop测定法确定蛋白质浓度 注意:可以使用任何确定蛋白质浓度的方法。
    10. 将100μl等分的蛋白质分配到1.5ml预先置入的微量离心管中
    11. 加入25μl甘油(最终20%)
    12. 将蛋白质储存在-80°C直到需要。


      图1.纯化的细菌蛋白激酶的实例使用来自嗜肺军团菌细菌GST-LegK2的过表达和纯化的细菌蛋白激酶的SDS-PAGE凝胶的考马斯染色。 MW:分子量,NI:细菌的非诱导级分,I:细菌的诱导级分,E:洗脱级分
  3. 净化ARP2/3复合物
    1. 所有试剂和纯化的ARP2/3复合物都必须在4°C下进行
    2. 将含有VCA偶联的琼脂糖凝胶的1ml聚丙烯柱与缓冲液A平衡
    3. 在4℃下将Acanthamoeba castellanii 的肌动蛋白耗尽的提取物应用于该柱。
      注意:如前所述在Zuchero(2007)中制备的棘皮棘球绦虫肌动蛋白耗尽的提取物。 Acanthamoeba体外肌动蛋白装配测定和纯化。方法Mol Biol 370:213-226。
    4. 用20个柱体积的缓冲液A洗涤
    5. 用ARP2/3洗脱缓冲液洗脱ARP2/3
    6. 通过在洗脱缓冲液中平衡的1ml苯基Sepharose柱,然后通过1.5柱体积的洗脱缓冲液,将ARP2/3浓缩的级分通过。
    7. 收集滤液,并通过SDS-PAGE鉴定富含ARP2/3的级分
    8. 使用PD10色谱柱或透析将ARP2/3复合物浓缩物转化为ARP2/3存储缓冲液。
    9. 等分的ARP2/3复合物,在液体N 2中闪光冷冻并存储在-80℃。


      图2.纯化的ARP2/3复合物使用从Acanthamoeba castellanii纯化的ARP2/3复合物的SDS-PAGE凝胶的考马斯染色。 MW:分子量
  4. ARP2/3复合物的脱磷酸酯
    1. 用2mM Tris pH8.0稀释ARP2/3复合物1:1,并与含有1U南极磷酸酶的HipH缓冲液结合。
    2. 在30℃孵育1.5小时。
    3. 模拟处理的对照物应与65℃的热灭活磷酸酶孵育30分钟。
    4. 亲和纯化使用N-WASP VCA偶联至活化的CH-Sepharose的去磷酸化的ARP2/3复合物。
    5. 用ARP2/3洗脱缓冲液从Sepharose中洗脱ARP2/3复合物。
    6. 缓冲液A透析ARP2/3在4°C过夜。

  5. 体外磷酸化测定
    1. 在1.5ml微量管中准备以下反应:

      表1.磷酸化测定的反应


    2. 在37℃下在水浴中孵育反应30分钟。
    3. 在磷酸酶试管中,加入5μl5x HipH缓冲液,加入2μl南极磷酸酶,浓度为5000 U/ml(最终10 U),并在30°C水浴中再孵育1 h。
    4. 通过加入5μl5x Laemmli加载缓冲液停止反应。

  6. 用抗磷酸腺嘌呤抗体通过蛋白质印迹检测磷酸化水平
    1. 以12%准备两种SDS-PAGE凝胶。
    2. 在干燥加热器中将样品在100℃下加热5分钟。
    3. 加入20μl加热的样品和5μl蛋白质梯度凝胶,并在SDS-PAGE迁移缓冲液中以35mA电泳,凝胶电泳约45分钟。
      注意:迁移的电流和时间取决于凝胶尺寸。
    4. 在陪替氏培养皿中用考马斯蓝染色其中一个凝胶。
      1. 在轻轻搅动下,在染色溶液中将凝胶染色20分钟
      2. 将凝胶洗涤30分钟至1小时,同时在脱色溶液中轻轻摇动
      3. 分析不同的分数。
    5. 用第二个凝胶进行蛋白质印迹。
      1. 在半干燥条件下将蛋白质从第二个凝胶转移到硝酸纤维素膜上,持续1小时。
      2. 将膜放入适合搅拌的盘子或锥形管中。膜与蛋白质的一侧不能与容器接触,溶液必须完全覆盖膜。
      3. 在TBS-5%BSA中温和旋转,在室温下封闭膜1小时
      4. 在TBS-0.1%吐温20中洗涤膜3×5分钟
      5. 在抗磷酸苏氨酸抗体溶液中,在室温下温和旋转膜孵育1小时。
        注意:按照本协议,我们检测苏氨酸磷酸化水平。还可以使用抗磷酸酪氨酸抗体(EMD Millipore,目录号:AB1607)或另一种抗磷酸酪氨酸抗体检测ARP2/3复合物的酪氨酸磷酸化水平,如有必要,调整程序以遵循制造商的建议。 br />
      6. 在TBS-0.1%吐温20中洗涤膜3×5分钟
      7. 在室温下孵育1小时,在抗小鼠过氧化物酶抗体溶液中温和旋转
      8. 在TBS-0.1%吐温20中洗涤膜3×5分钟
      9. 用TBS清洗膜。
      10. 用SuperSignal West Pico Chemiluminescent Substrate Kit检测抗磷酸化苏氨酸标记。
        1. 将1毫升化学发光底物(500微升的稳定的过氧化物溶液+ 500微升的鲁米诺/增强剂溶液)放在一片石蜡膜上。
        2. 将膜直接置于化学发光底物上,使蛋白质与过氧化物酶底物直接接触 注意:注意,清除膜和基材之间存在的任何气泡。
        3. 在黑暗中孵育5分钟。
        4. 将溶液从膜上排出并放在支架上
        5. 用塑料包裹膜,以防止干燥。
        6. 使用ChemiStart或适当的检测系统在不同曝光下的图像膜。
        注意:
        1. 可以根据设备制造商的建议使用用于检测化学发光信号的其他方法。
        2. 您也可以检测蛋白激酶的自磷酸化状态。然而,我们建议如果激酶及其底物的分子量完全不同,则以不同的聚丙烯酰胺浓度进行分析。
        3. 描述ARP2/3磷酸化的原始出版物也可以帮助设定条件和分析:LeClaire,LL 3 rd ,Baumgartner,M.,Iwasa,JH,Mullins,RD,Barber,DL(2008)。 Arp2/3复合物的磷酸化对于使肌动蛋白丝成核是必需的。 J Cell Biol 182(4):647-54。

笔记

  1. 为了优化细菌蛋白质的表达和纯化,可以在SDS-PAGE后通过考马斯蓝染色分析对应于每个步骤的所有级分。
  2. 步骤A13的细菌颗粒可以在-80℃冷冻。
  3. 对于我们的ARP2/3磷酸化测定,我们使用Sigma的抗磷酸核糖体抗体。然而,我们发现抗体标记是不一致的,需要优化实验条件。为了测试磷酸化活性,我们发现来自Cell Signaling(#9381)的多克隆磷酸苏氨酸抗体显示出更一致的标记模式,并且更具体地用于标记。
  4. 蛋白印迹分析的几个步骤可以进行一夜;转移(0.2mA/cm 2);阻塞(4℃);或与第一抗体孵育(4℃)
  5. 戴手套来保护样品免受蛋白酶和/或污染蛋白质的影响

食谱

  1. LB培养基+氨苄青霉素(100μg/ml)+卡那霉素(25μg/ml)
    0.2 ml 100mg/ml氨苄青霉素储备液
    25μl50 mg/ml卡那霉素储备液
    200毫升LB培养基
    1. LB培养基
      20克LB培养基(Lennox)
      1L的dH 2 O
      等份由200毫升
      通过高压消毒灭菌
    2. 100毫克/毫升氨苄青霉素储备溶液
      5克氨苄青霉素
      50ml dH 2 O
      等待并冻结解决方案直到需要
    3. 50毫克/毫升卡那霉素储备溶液
      2.5克卡那霉素
      50ml dH 2 O
      等待并冻结解决方案直到需要
  2. 0.1 M IPTG
    238毫克IPTG
    10ml dH 2 O
    等待并冻结直到需要
  3. 10x PBS(1升)
    80克NaCl
    2克KCl
    14.4g Na 2 HPO 4
    2.4g KH 2 PO 4
    将pH调节至7.4
    将dH 2 O添加到1 L
  4. 1x PBS(1升)
    100毫升10倍PBS
    900毫升的dH 2 O -/-
  5. GST洗脱缓冲液
    50 mM Tris HCl pH 8.0
    10 mM谷胱甘肽
  6. 透析缓冲液
    50 mM Tris HCl pH 7.5
    150 mM NaCl
    10%甘油
  7. ARP2/3洗脱缓冲液
    10mM Tris,pH 8.0
    400mM MgCl 2
  8. ARP2/3存储缓冲区
    1 mM HEPES
    1 mM DTT
    10%甘油
    调节pH至7.0
  9. 10x磷酸化缓冲液
    250mM Tris HCl pH 7.5
    50mM MnCl 2
    50 mM DTT
  10. ATP溶液为0.5mg/ml 1毫克ATP
    2ml dH 2 O
  11. 5x HipH缓冲区
    50mM Tris pH 8.0
    1mM Mg 2 Cl 2
    0.1mM ZnCl 2
  12. 缓冲区A 10x解决方案
    500 mM KCl
    20mM MgCl 2
    10 mM ATP
    100mM Tris,pH7.5
  13. 5x Laemmli加载缓冲区
    5 ml 10%SDS
    6.25ml 0.5M Tris HCl pH6.8
    5毫升甘油
    7.5ml dH 2 O
    1次溴酚蓝
    2.5μl1 Mβ-巯基乙醇
  14. 12%SDS-PAGE凝胶(2微型凝胶)
    1. 堆积凝胶
      1ml 30%ProtoGel(30%丙烯酰胺,0.8%双丙烯酰胺)
      1.25ml 0.5M Tris HCl pH6.8
      2.65ml dH 2 O 50μl10%SDS
      50μl10%APS
      5μlTEMED
    2. 分离凝胶
      4ml 30%ProtoGel(30%丙烯酰胺,0.8%双丙烯酰胺)
      2.5ml 1.5M Tris HCl pH8.8
      3ml dH 2 O O// 100μl10%SDS
      100μl10%APS
      10μlTEMED
  15. 迁移缓冲区
    25 mM Tris
    192mM甘氨酸
    0.1%SDS
  16. 染色溶液
    10%冰醋酸
    40%乙醇
    0.04%考马斯亮蓝R-250
  17. 解决方案
    10%冰醋酸
    5%乙醇
  18. 转移解决方案1
    300 mM Tris
    20%甲醇
  19. 转移解决方案2
    25 mM Tris
    20%甲醇
  20. 转移解决方案3
    25 mM Tris
    40mM 6-氨基己酸
    20%甲醇
  21. 组装半干运输
    阴极( - ) 

    阳极(+)
  22. TBS
    100 mM Tris
    150 mM NaCl
    将pH调节至8
  23. TBS-5%BSA
    0.5克BSA
    10毫升TBS
  24. TBS-0.1%吐温20
    1ml吐温20
    1 L TBS
  25. 抗磷酸核糖抗体溶液
    PBS-5%BSA-0.1%吐温20
    单克隆小鼠抗磷酸苏氨酸抗体(1/500)
  26. 抗小鼠过氧化物酶抗体溶液
    TBS-5%BSA-0.1%吐温20
    山羊抗小鼠IgG过氧化物酶抗体(1/5,000)

致谢

这项工作是在里昂大学的LABEX ECOFECT(ANR-11-LABX-0042)的框架内进行的,由法国国家研究机构(ANR-11-IDEX-0007)运营的计划投资项目(ANR-11-IDEX-0007) ANR)。这项工作由国家科技中心(UMR 5308),国立雷神学院(U1111)和里昂大学1所资助。
该协议源自以前描述的协议:(Hervet等人,2011; Zuchero,2007和LeClaire等人,2008)

参考文献

  1. Amrein,KE,Takacs,B.,Stieger,M.,Molnos,J.,Flint,NA和Burn,P(1995)。< a class ="ke-insertfile"href ="http: .ncbi.nlm.nih.gov/pubmed/7862631"target ="_ blank">来自大肠杆菌表达系统的重组人p50csk蛋白质 - 酪氨酸激酶的纯化和表征,其过量产生细菌分子伴侣GroES和GroEL 。 Proc Natl Acad Sci USA 92(4):1048-1052。
  2. Haglund,CM和Welch,MD(2011)。病原体和聚合物:微生物 - 宿主相互作用照射细胞骨架。细胞生物学 195(1):7-17。
  3. Hervet,E.,Charpentier,X.,Vianney,A.,Lazzaroni,JC,Gilbert,C.,Atlan,D。和Doublet,P。(2011)。蛋白激酶LegK2是涉及内质网募集和肺炎衣原体细胞内复制的IV型分泌系统效应子,/em>。 Infect Immun 79(5):1936-50。
  4. Leclaire,LL,3rd,Baumgartner,M.,Iwasa,JH,Mullins,RD and Barber,DL(2008)。  Arp2/3复合物的磷酸化对于使肌动蛋白丝成核是必需的.Jell Cell Biol.182(4):647-654 。
  5. LeClaire,LL,Rana,M.,Baumgartner,M.and Barber,DL(2015)。  Nck相互作用的激酶NIK通过磷酸化Arp2亚基而增加Arp2/3复合物活性。 208(2):161-170。
  6. Michard,C.,Sperandio,D.,Bailo,N.,Pizarro-Cerda,J.,LeClaire,L.,Chadeau-Argaud,E.,Pombo-Gregoire,I.,Hervet,E.,Vianney, ,Gilbert,C.,Faure,M.,Cossart,P。和Doublet,P。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih。 gov/pubmed/25944859"target ="_ blank">军团菌激酶LegK2靶向ARP2/3复合物,以抑制吞噬体上的肌动蛋白成核,并允许细菌逃避晚期内吞途径。 6(3):e00354-00315。
  7. Narayanan,A.,LeClaire,LL,3rd,Barber,DL和Jacobson,MP(2011)。  Arp2亚基的磷酸化可以缓解Arp2/3复合物激活的自身抑制相互作用。 PLoS Comput Biol 7(11):e1002226。 />
  8. Smith,DB和Johnson,KS(1988)。  Single - 在大肠杆菌中表达的多肽纯化作为与谷胱甘肽S-转移酶的融合物。基因67(1):31-40。
  9. Vadlamudi,RK,Li,F.,Barnes,CJ,Bagheri-Yarmand,R.和Kumar,R。(2004)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/14749719"target ="_ blank"> p41-人类Arp2/3复合体的弧亚基是p21激活的激酶-1相互作用的底物。 EMBO Rep 5(2):154-160。
  10. Welch,MD和Way,M.(2013)。 Arp2/3介导的基于肌动蛋白的动力:病原体滥用的尾巴。细胞宿主微生物 14(3):242-255。
  11. Zuchero,JB(2007)。  体外肌动蛋白组装测定和从Acanthamoeba 纯化。方法Mol Biol 370:213-226。
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引用:Michard, C., LeClaire, L. L. and Doublet, P. (2017). ARP2/3 Phosphorylation Assay in the Presence of Recombinant Bacterial Effectors. Bio-protocol 7(7): e2208. DOI: 10.21769/BioProtoc.2208.
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