Arrestin‐3‐Dependent Activation of c‐Jun N‐Terminal Kinases (JNKs)

Xuanzhi Zhan1, Seunghyi Kook1, Tamer S. Kaoud2, Kevin N. Dalby3, Eugenia V. Gurevich4, Vsevolod V. Gurevich4

1 These authors contributed equally to this work, 2 Faculty of Pharmacy, Minia University, Minia, 3 Division of Medicinal Chemistry, The University of Texas at Austin, Austin, Texas, 4 Department of Pharmacology, Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.12
DOI:  10.1002/0471141755.ph0212s68
Online Posting Date:  March, 2015
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Abstract

Only one out of four mammalian arrestin subtypes, arrestin‐3, facilitates the activation of JNK family kinases. Here we describe two different protocols used for elucidating the mechanisms involved. One is based on reconstitution of signaling modules from purified proteins: arrestin‐3, MKK4, MKK7, JNK1, JNK2, and JNK3. The main advantage of this method is that it unambiguously establishes which effects are direct because only intended purified proteins are present in these assays. The key drawback is that the upstream‐most kinases of these cascades, ASK1 or other MAPKKKs, are not available in purified form, limiting reconstitution to incomplete two‐kinase modules. The other approach is used for analyzing the effects of arrestin‐3 on JNK activation in intact cells. In this case, signaling modules include ASK1 and/or other MAPKKKs. However, as every cell expresses thousands of different proteins their possible effects on the readout cannot be excluded. Nonetheless, the combination of in vitro reconstitution from purified proteins and cell‐based assays makes it possible to elucidate the mechanisms of arrestin‐3‐dependent activation of JNK family kinases. © 2015 by John Wiley & Sons, Inc.

Keywords: c‐Jun N‐terminal kinase (JNK); scaffold; activation; arrestin; biphasic dependence

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Construction of Arrestin 3‐Scaffolded MKK4/7‐JNK1/2/3 Signaling Modules In Vitro Using Purified Proteins
  • Alternate Protocol 1: Arrestin‐3 Mediated JNK1/2 Activation by MKK4/7 (Measurements of JNK1/2 Phosphorylation Using Immunoblotting with Phospho‐JNK Antibodies)
  • Basic Protocol 2: The Role of Arrestin‐3 in JNK Activation in Intact Cells
  • Alternate Protocol 2: MKK4‐Induced JNK Activation
  • Basic Protocol 3: Biphasic Effect of Arrestin‐3 on ASK1‐ and MKK7‐Stimulated JNK Phosphorylation in Cells
  • Support Protocol 1: Expression, Purification, and Activation of GST‐MKK4
  • Support Protocol 2: Expression, Purification, and Activation of GST‐MKK7‐His6
  • Support Protocol 3: Expression, Purification, and Activation of Tag‐Less JNK1α1
  • Support Protocol 4: Expression, Purification and Activation of Tag‐Less JNK2α2
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Construction of Arrestin 3‐Scaffolded MKK4/7‐JNK1/2/3 Signaling Modules In Vitro Using Purified Proteins

  Materials
  • Purified proteins
  • Ice
  • Bradford Assay (Bio‐Rad)
  • Kinase mix (see Table 2.12.1)
  • Antibodies: phospho‐JNK antibody (rabbit, #9251, Cell Signaling Technology); Pan‐JNK antibody (rabbit, #9252, Cell Signaling Technology)
  • Arrestin‐3 solutions (see Table 2.12.2)
  • 10 mM ATP (solution in distilled water; Sigma, cat. no. A2383)
  • ATP, [γ‐32P] (PerkinElmer)
  • Sodium dodecyl sulfate (SDS) buffer (Sigma, cat. no. S3401)
  • 10% SDS‐PAGE gel ( appendix 3B; Gallagher and Sasse, )
  • Coomassie blueScintillation cocktail (Scintisafe Econo 2, Fisher)
  • Ultracentrifuge
  • 1.5 ml tubes
  • Vortex mixer
  • 30°C water bath
  • Gel dryer (e.g., Bio‐Rad)
  • Autoradiography cassette
  • Scintillation vials
  • Scintillation counter
  • Shaking platform (room temperature)
  • Additional reagents and equipment for Protein Analysis by SDS‐PAGE and Detection by Coomassie Blue or Silver Staining (see Gallagher and Sasse, )
Table 2.2.1   MaterialsPreparing the Kinase Mix aPreparing Arrestin‐3 Solutions

Volume (each) Volume (20×)
10× Kinase assay buffer (see recipe) 2 μl 40 μl
100 mM DTT 0.4 μl 8 μl
JNK3α2 (12 μM stock) 0.8 μl 16 μl
p‐MKK4 or p‐MKK7 (10 μM stocks) 0.1 μl 2 μl
Total 3.3 μl 66 μl
Tube Number A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13
Arrestin‐3 concentration (μM) 0 0.1 0.2 0.4 0.6 1.2 2 4 8 12 20 30 45
Arrestin‐3 (μl) 0 2 a 4 8 12 2.4 4 8 2.4 3.6 6 9.2 13.2
Tris buffer (μl) b 14.7 12.7 10.7 6.7 2.7 12.3 10.7 6.7 12.3 11.1 8.7 5.5 1.5

 aAntibodies: phospho‐JNK antibody (rabbit, Cell Signaling Technology, cat. no. 9251); total JNK antibody (rabbit, Cell Signaling Technology, cat. no. 9252).
Table 2.2.2   MaterialsPreparing the Kinase Mix aPreparing Arrestin‐3 Solutions

Volume (each) Volume (20×)
10× Kinase assay buffer (see recipe) 2 μl 40 μl
100 mM DTT 0.4 μl 8 μl
JNK3α2 (12 μM stock) 0.8 μl 16 μl
p‐MKK4 or p‐MKK7 (10 μM stocks) 0.1 μl 2 μl
Total 3.3 μl 66 μl
Tube Number A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13
Arrestin‐3 concentration (μM) 0 0.1 0.2 0.4 0.6 1.2 2 4 8 12 20 30 45
Arrestin‐3 (μl) 0 2 a 4 8 12 2.4 4 8 2.4 3.6 6 9.2 13.2
Tris buffer (μl) b 14.7 12.7 10.7 6.7 2.7 12.3 10.7 6.7 12.3 11.1 8.7 5.5 1.5

 aArrestin stock concentrations: For A2‐A5, 1 μM; For A6‐A8, 10 μM; For A9‐A13, 65 μM.
 bTris buffer: 10 mM Tris·Cl, pH 7.5, and 100 mM NaCl.

Alternate Protocol 1: Arrestin‐3 Mediated JNK1/2 Activation by MKK4/7 (Measurements of JNK1/2 Phosphorylation Using Immunoblotting with Phospho‐JNK Antibodies)

  Additional Materials (also see protocol 1)
  • PVDF membrane (Millipore)
  • Enhanced chemiluminescence reagent (e.g., SuperSignal West Pico from Pierce)
  • X‐ray film
  • Quantity One software (Bio‐Rad)

Basic Protocol 2: The Role of Arrestin‐3 in JNK Activation in Intact Cells

  Materials
  • COS‐7 (African green monkey fibroblast) cells (ATCC)
  • Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/liter glucose, L‐glutamine and sodium pyruvate (CORNING cellgro, cat. no. 10‐013‐CV)
  • Fetal bovine serum, Qualified (Life Technologies, cat. no. 2017‐07)
  • Penicillin/Streptomycin (Life Technologies, cat. no. 15140‐122)
  • Opti‐MEM I Reduced Serum Medium, no phenol red (Invitrogen, cat. no. 11058‐021)
  • pcDNA3‐HA‐AKS1 and pcDNA3
  • pcDNA3‐Arrestin‐3
  • FuGENE HD Transfection Reagent (Promega, cat. no. E231A)
  • Growth medium
  • Serum‐free medium
  • Ice
  • Dulbecco's Phosphate‐buffered saline (DPBS) without calcium and magnesium (CORNING cellgro, cat. no. 21‐031‐CV)
  • Lysis buffer (see recipe)
  • Bio‐Rad Protein Assay Dye Reagent Concentrate (Bio‐Rad, cat. no. 500‐0006)
  • Antibodies for immunoblot:
    • F4C1: Mouse monoclonal antibody that detects the epitope DGVVLVD, a sequence that is present in all known mammalian arrestins (Donoso et al., ) or F431: Rabbit polyclonal antibody that detects the same epitope as F4C1 (Song et al., )
    • Phospho‐SAPK/JNK (Thr183/Tyr185) antibody (Cell Signaling Technology, cat . no. 9251), SAPK/JNK antibody (Cell Signaling Technology, cat. no. 9252), HA‐Tag (6E2) Mouse antibody (Cell Signaling Technology, cat. no. 2367), Monoclonal anti‐Flag M2 antibody (Sigma, cat. no. F3165)
  • 2× SDS Sample buffer (Sigma)
  • 8% SDS‐PAGE gel
  • 0.05% Trypsin‐EDTA (Life Technologies, cat. no. 25300‐054)
  • 37°C humidified incubator with 5% CO 2
  • 6‐well plates
  • 1.5‐ml microcentrifuge tubes
  • Cell scraper
  • 95°C heat block (Isotemp 125D, Fisher Scientific)
  • Electrophoresis equipment
  • PVDF membrane (Millipore)
  • Additional reagents and the equipment needed for performing SDS‐PAGE electrophoresis ( appendix 3B; Gallagher and Sasse, ), immunoblotting (described in Zhan et al., ; Zhan et al., )., and measuring the protein concentration ( appendix 3A; Olson and Markwell, )
NOTE: Add all protease inhibitors immediately before use, especially PMSF, which is unstable in aqueous solutions.

Alternate Protocol 2: MKK4‐Induced JNK Activation

  Materials
  • Plasmid pGEX4T1‐MKK4 vector is a construct encoding full‐length wild type Homo sapiens mitogen‐activated protein kinase kinase 4 (MAP2K4) (GenBank accession number NM_003010) with an N‐terminal‐cleavable GST–tag (Yan et al., ; Zhan et al., ); this plasmid is available upon request from Dr. K.N. Dalby (Division of Medicinal Chemistry; BME 6.202B; 1 University Station C0850, Austin, Texas 78712‐1074; E‐mail: )
  • BL21 (DE3) (Novagen): prepare electrocompetent cells as described previously (see Gonzales et al., )
  • Luria Broth (LB) medium (Sigma) containing 50 μg/ml ampicillin sodium (Sigma)
  • Terrific Broth (TB) medium (Sigma) containing 50 μg/ml ampicillin sodium (Sigma)
  • 1 M Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) solution in sterile water (Sigma)
  • Liquid nitrogen
  • Buffer A (see recipe)
  • 1 M MgCl solution (Sigma)
  • Lysozyme lyophilized enzyme (MP Biomedicals)
  • 100% (v/v) Glycerol
  • 10% (v/v) Triton in water (Sigma)
  • Glutathione Sepharose High Performance (Amersham Biosciences); on the day of the experiment, pack the required beads in a glass Econo‐Column, wash and equilibrate in buffer A
  • Buffer B (see recipe)
  • Bradford reagent (Bio‐Rad)
  • GST‐MEKK1c (C terminal 320 amino acids corresponding to the catalytic domain) is necessary for activating GST‐MKK4, GST‐MKK7‐His 6 [the protocol for GST‐MEKK1c vector construction and protein purification has been described previously (see Khokhlatchev et al., ; Gallagher et al., ); this plasmid is available upon request from Dr. K.N. Dalby (Division of Medicinal Chemistry; BME 6.202B; 1 University Station C0850, Austin, Texas 78712‐1074; E‐mail: )
  • 50 mM ATP (Roche) in 25 mM HEPES buffer (pH 7.5)
  • Buffer C (see recipe)
  • Buffer D (see recipe)
  • Shaking incubator
  • Sonicator
  • Econo‐Column Chromatography glass columns, 2.5 × 20 cm (98 ml) (Bio‐Rad)
  • Amicon Ultra‐15 concentrator
  • Gel filtration column: 120 ml HiLoad 16/60 Superdex 200 prep grade column (Amersham)
  • Additional reagents and equipment for transforming the pGEX4T1‐MKK4 vector into BL21 (DE3) electrocompetent cells (House et al., ) and measuring the protein concentration (Olson and Markwell, )

Basic Protocol 3: Biphasic Effect of Arrestin‐3 on ASK1‐ and MKK7‐Stimulated JNK Phosphorylation in Cells

  Additional Materials (also see protocol 6)
  • Plasmid: pGEX‐MKK7‐His 6 vector is a construct encoding full length, wild‐type Mus musculus mitogen‐activated protein kinase kinase 7 (GenBank accession number NM_011944) with N‐terminal cleavable GST–tag and C‐terminal cleavable His‐tag (Madsen et al., ; Zhan et al., ); this plasmid is available upon request from Dr. K.N. Dalby (Division of Medicinal Chemistry; BME 6.202B; 1 University Station C0850; Austin, Texas 78712‐1074; E‐mail: )
  • Buffer E (see recipe)
  • NaCl (Biological grade MP Biomedical)
  • Ni‐NTA beads (Qiagen); on the day of the experiment, pack the required beads in a glass Econo‐Column, wash and, equilibrate in buffer E

Support Protocol 1: Expression, Purification, and Activation of GST‐MKK4

  Additional Materials (also see Support Protocols protocol 61 and protocol 72)
  • Plasmid pET28a(+)Tev‐JNK1α1 vector is a construct encoding full length wild‐type Homo sapien mitogen‐activated protein kinase 8 (GenBank accession number NM_002750) with N‐terminal Tev (Tobacco Etch Virus ) cleavable His‐tag (Yan et al., ); this plasmid is available upon request from Dr. K.N. Dalby (Division of Medicinal Chemistry; BME 6.202B; 1 University Station C0850; Austin, Texas 78712‐1074; E‐mail: )
  • Luria Broth (LB) medium containing 30 μg/ml kanamycin (Sigma)
  • Imidazole
  • Buffer F (see recipe)
  • Buffer G (see recipe)
  • TEV (Tobacco Etch Virus ) protease[Express and purify TEV protease as previously described (Abramczyk et al., )]; plasmid is available in Addgene, plasmid 8827: pRK793, TEV protease, S219V mutant
  • CaCl 2
  • Mono Q HR 10/10 anion‐exchange column (Amersham)
  • HiPrep 26/10 desalting column (Amersham)

Support Protocol 2: Expression, Purification, and Activation of GST‐MKK7‐His6

  Additional Materials (also see Support Protocols protocol 61, protocol 72, and protocol 83)
  • Plasmid pET28a(+)‐JNK2α2 vector is a construct encoding full‐length wild‐type Homo sapien mitogen‐activated protein kinase 9 (GenBank accession number NM_002752) with N‐terminal Thr (Thrombin) cleavable His‐tag (Madsen et al., ); this plasmid is available upon request from Dr. K.N. Dalby (Division of Medicinal Chemistry; BME 6.202B; 1 University Station C0850; Austin, Texas 78712‐1074; E‐mail: )
  • Thrombin protease (Novagen)
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Literature Cited

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