重组细菌效应子存在条件下ARP2/3磷酸化修饰的检测

Céline  Michard; Lawrence  L.  LeClaire; Patricia  Doublet

doi:10.21769/BioProtoc.2208

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Peer-reviewed

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

Céline Michard email

LL Lawrence L. LeClaire

PD Patricia Doublet email

发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2208 浏览次数: 8093

评审: Yurong XieTatsuki KunohAnonymous reviewer(s)

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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

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)
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)
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)
Dephosphorylation of ARP2/3 complex
1. Microtubes (1.5 ml)
2. 3,500 MWCO Mini dialysis units (Thermo Fisher Scientific, Thermo Scientific^TM, 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)
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)
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 Ruler^TM prestained protein ladder (Thermo Fisher Scientific, Thermo Scientific^TM, 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 Scientific^TM, 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

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

Procedure

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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.