Analyzing Mitogen‐Activated Protein Kinase (MAPK) Activities in T Cells

Alan J. Whitmarsh1, Roger J. Davis2

1 University of Manchester, School of Biological Sciences, Manchester, 2 University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 11.8
DOI:  10.1002/0471142735.im1108s58
Online Posting Date:  February, 2004
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Abstract

The recognition of peptide antigens by T cells through their antigen receptors (T cell receptors, or TCR), together with the ligation of additional surface molecules called costimulatory receptors, rapidly induces interactive signaling pathways that lead to transcriptional initiation at genes such as that of the autocrine growth factor interleukin 2 (IL‐2). Activation of the ERK and JNK subfamilies of MAPK mediates some of these signals. This unit presents procedures for a solid‐phase kinase assay and immune‐complex kinase assay to measure JNK and ERK activities, respectively, in T cells that have been appropriately stimulated. Also described is a procedure for preparing GSThyphen;cJun/GSH‐Sepharose beads needed in the solid‐phase JNK protein kinase activity assay.

     
 
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Table of Contents

  • Basic Protocol 1: Immune Complex Protein Kinase Assay
  • Basic Protocol 2: Solid‐Phase Protein Kinase Assay for JNK Activity
  • Basic Protocol 3: In Situ Detection of JNK Protein Kinase Activity Following SDS‐PAGE (“In‐Gel” Kinase Assay)
  • Basic Protocol 4: Measuring MAPK Activation Using Phosphorylation Site–Specific Antibodies
  • Support Protocol 1: Preparation of GST‐MAPK Substrate Fusion Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Immune Complex Protein Kinase Assay

  Materials
  • Mouse T cells (0.5–1.0 × 106 cells per assay; see units 3.1 3.4 for isolation techniques)
  • Triton lysis buffer (TLB; see recipe), ice cold
  • Anti‐MAPK polyclonal antibodies (all from Santa Cruz Biotechnology): for ERK2, C‐14; for JNK1, C‐17); for p38, C‐20
  • 50% protein A–Sepharose slurry (see recipe)
  • Kinase buffer (KB; see recipe)
  • 2 mg/ml GST‐MAPK substrate (prepared as described in protocol 5): GST‐Elk1(310‐428), GST‐cJun(1‐79), or GST‐ATF2(1‐109)
  • 1 mM ATP (Sigma; store at −20°C in aliquots)
  • 10 mCi/ml [γ‐32P]ATP (6000 Ci/mmol; Amersham Pharmacia Biotech)
  • 4× SDS gel loading buffer (see recipe)
  • 12% SDS‐PAGE minigel (unit 8.4)
  • Coomassie stain (see recipe)
  • Destaining solution (see recipe)
  • Refrigerated centrifuge
  • Rotating platform mixer
  • 30° and 100°C heating blocks
  • Staining dishes to accommodate minigels
  • Whatman 3MM filter paper
  • Additional reagents and equipment for SDS‐PAGE using minigels (unit 8.4) and detection/quantitation of proteins via autoradiography and phosphor imaging ( appendix 3J)

Basic Protocol 2: Solid‐Phase Protein Kinase Assay for JNK Activity

  Materials
  • Mouse T cells (0.5–1.0 × 106 cells per assay; see units 3.1 3.4 for isolation techniques)
  • Triton lysis buffer (TLB; see recipe), ice cold
  • 50% (v/v) suspension of GST‐cJun(1‐79)/GSH‐Sepharose beads in PBS (prepare as described in protocol 5, steps to ; omit subsequent elution steps)
  • Kinase buffer (KB; see recipe)
  • 1 mM ATP (Sigma; store at −20°C in aliquots)
  • 10 mCi/ml [γ‐32P]ATP (6000 Ci/mmol; Amersham Pharmacia Biotech)
  • 4× SDS gel loading buffer (see recipe)
  • 12% SDS‐PAGE minigel (unit 8.4)
  • Coomassie stain (see recipe)
  • Destaining solution (see recipe)
  • Refrigerated centrifuge
  • Rotating platform mixer
  • 30° and 100°C heating blocks
  • Staining dishes to accommodate minigels
  • Whatman 3MM filter paper
  • Additional reagents and equipment for SDS‐PAGE using minigels (unit 8.4) and detection/quantitation of proteins via autoradiography and phosphor imaging ( appendix 3J)

Basic Protocol 3: In Situ Detection of JNK Protein Kinase Activity Following SDS‐PAGE (“In‐Gel” Kinase Assay)

  Materials
  • Mouse T cells (∼0.5 × 106 cells per assay; see units 3.1 3.4 for isolation techniques)
  • Triton lysis buffer (TLB; see recipe), ice cold
  • GST (control) or GST‐cJun(1‐79) (prepared as described in protocol 5)
  • 4× SDS gel loading buffer (see recipe)
  • 20% (v/v) isopropanol in 50 mM Tris·Cl, pH 8.0 (see appendix 2A for the Tris buffer)
  • Buffer A (see recipe)
  • 6 M guanidine·HCl in buffer A, filtered through a 0.45‐µm filter just prior to use
  • 0.04% (v/v) Tween 40 (Sigma) in buffer A
  • Buffer B (see recipe)
  • 1 mM ATP (Sigma; store at −20°C in aliquots)
  • 10 mCi/ml [γ‐32P]ATP (6000 Ci/mmol; Amersham Pharmacia Biotech)
  • 5% (w/v) trichloroacetic acid (TCA)/1% (w/v) sodium pyrophosphate
  • Refrigerated centrifuge
  • 100°C heating block
  • Rocking platform mixer
  • Staining trays to accommodate minigels
  • Geiger counter
  • Whatman 3MM filter paper
  • Additional reagents and equipment for SDS‐PAGE using minigels (unit 8.4) and detection/quantitation of proteins via autoradiography and phosphor imaging ( appendix 3J)

Basic Protocol 4: Measuring MAPK Activation Using Phosphorylation Site–Specific Antibodies

  Materials
  • Mouse T cells (1.0 × 106 cells per assay; see units 3.1 3.4 for isolation techniques)
  • Triton lysis buffer (TLB; see recipe), ice cold
  • 4× SDS gel loading buffer (see recipe)
  • 12% SDS‐PAGE minigel (unit 8.4)
  • Methanol
  • Western transfer buffer (WTB; see recipe)
  • 5% (w/v) bovine serum albumin (BSA) in western blotting buffer (WBB; see recipe)
  • Primary antibody: rabbit polyclonal Phospho‐SAPK/JNK (Thr‐183/Tyr‐185) (Cell Signaling Technology)
  • 0.5% (v/v) Tween 20 in WBB (see recipe for WBB)
  • Secondary antibody: HRP‐conjugated anti–rabbit Ig (Amersham Pharmacia Biotech)
  • Western blotting buffer (WBB; see recipe)
  • Refrigerated centrifuge
  • 100°C heating block
  • Immobilon‐P transfer membrane (Millipore)
  • Gel blot paper (Schleicher & Schuell)
  • Semidry western transfer apparatus (Hoefer Scientific; also see unit 8.10)
  • Rocking platform mixer
  • X‐ray film (also see appendix 3J)
  • Additional reagents and equipment for SDS‐PAGE using minigels (unit 8.4) and immunoblotting and detection with chemiluminescent substrates (unit 8.10)

Support Protocol 1: Preparation of GST‐MAPK Substrate Fusion Proteins

  Materials
  • Transformation‐competent E. coli (BL21; Novagen)
  • Plasmid DNA encoding GST‐MAPK substrate fusion proteins (see discussion above)
  • LB medium (unit 10.3) containing 100 µg/ml ampicillin (Sigma; add from 100 mg/ml stock in H 2O, stored in aliquots at −20°C)
  • 100 mM isopropyl‐β‐D‐thiogalactopyranoside (IPTG; Promega; store in aliquots at −20°C)
  • Buffer X (see recipe) with and without protease inhibitors, ice‐cold
  • Triton X‐100 (Sigma)
  • Glutathione (GSH)‐Sepharose 4B (Amersham‐Pharmacia)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Protein concentration assay kit (Bio‐Rad)
  • 100 mM HEPES, pH 8.0
  • 50 mM glutathione (Sigma)/100 mM HEPES, pH 8.0
  • Buffer D (see recipe)
  • Liquid nitrogen or dry ice
  • 250‐ml and 2‐liter conical flasks
  • Shaking bacterial incubator
  • Spectrophotometer
  • Refrigerated centrifuge
  • Probe sonicator (e.g., Branson)
  • 50‐ml conical centrifuge tubes
  • Rotating mixer
  • 2‐ml polypropylene chromatography column (Qiagen)
  • 12‐kDa MWCO dialysis membrane
  • Additional reagents and equipment for introducing plasmid DNA into E. coli via CaCl 2/heat shock (Sambrook et al., ) or electroporation (unit 10.15), and for SDS‐PAGE (unit 8.4), Coomassie staining of gels (unit 8.9), and dialysis ( appendix 3H)
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Figures

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Literature Cited

Literature Cited
   Alberola‐Ila, J., Forbush, K.A., Seger, R., Krebs, E.G., and Perlmutter, R.M. 1995. Selective requirement for MAP kinase activation in thymocyte differentiation. Nature 373:620‐623.
   Bou, G., Villasis‐Keever, A., and Paya, C.V. 2003. Detection of JNK and p38 activation by flow cytometry analysis. Anal. Biochem. 317:147‐155.
   Conze, D., Krahl, T., Kennedy, N., Weiss, L., Lumsden, J., Hess, P., Flavell, R.A., Le Gros, G., Davis, R.J., and Rincon, M. 2002. c‐Jun NH2‐terminal kinase (JNK) 1 and JNK2 have distinct roles in CD8+ T cell activation. J. Exp. Med. 195:811‐823.
   Crompton, T., Gilmour, K.C., and Owen, M.J. 1996. The MAP kinase pathway controls differentiation from double‐negative to double‐positive thymocytes. Cell 86:243‐251.
   Diehl, N.L., Enslen, H., Fortner, K.A., Merritt, C., Stetson, N., Charland, C., Flavell, R.A., Davis, R.J., and Rincon, M. 2000. Activation of the p38 MAP kinase pathway arrests cell cycle progression and differentiation of immature thymocytes in vivo. J. Exp. Med. 191:139‐146.
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   Gotoh, Y., Nishida, E., Yamashita, T., Hoshi, M., Kawakami, M., and Sakai, H. 1990. Microtubule‐associated‐protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells: Identity with the mitogen‐activated MAP kinase of fibroblastic cells. Eur. J. Biochem. 193:661‐669.
   Gupta, S., Barrett, T., Whitmarsh, A.J., Cavanagh, J., Sluss, H.K., Dérijard, B., and Davis, R.J. 1996. Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO J. 15:2760‐2770.
   Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. 1993. Identification of an oncoprotein‐ and UV‐responsive protein kinase that binds and potentiates the c‐Jun activation domain. Genes & Dev. 7:2135‐2148.
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   Merritt, C., Emslen, H., Diehl, N.L., Conze, D., Davis, R.J., and Rincon, M. 2000. Activation of the p38 MAP kinase in vivo selectively induces apoptosis of CD8+ but not CD4+ T cells. Mol. Cell. Biol. 20:936‐946.
   Rincon, M., Whitmarsh, A.J., Yang, D.D., Weiss, L., Derijard, B., Jayaraj, P., Davis, R.J., and Flavell, R.A. 1998a. The JNK pathway regulates the in vivo deletion of immature CD4+CD8+ thymocytes. J. Exp. Med. 188:1817‐1830.
   Rincon, M., Enslen, H., Raingeaud, J., Recht, M., Zapton, T., Su, M.S‐S., Penix, L.A., Davis, R.J., and Flavell, R.A. 1998b. Interferon‐γ expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway. EMBO J. 17:2817–2829.
   Rincon, M., Conze, D., Weiss, L., Diehl, N.L., Fortner, K.A., Yang, D.D., Flavell, R.A., Enslen, H., Whitmarsh, A.J., and Davis, R.J. 2000. Do T cells care about the mitogen‐activated protein kinase signaling pathways? Immunol. Cell Biol. 78:166–175.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual (2nd ed.) Cold Spring Harbor Press, Cold Spring Harbor, New York.
   Smith, D. and Johnson, K.S. 1988. Single‐step purification of polypeptides expressed in Escherichia coli as fusions with glutathione‐S‐transferase. Gene 67:31‐40.
   Su, B., Jacinto, E., Hibi, M., Kallunki, T., Karin, M., and Ben‐Neriah, Y. 1994. JNK is involved in signal integration during costimulation of T lymphocytes. Cell 77:727‐736.
   Sugawara, T., Moriguchi, T., Nishida, E., and Takahama, Y. 1998. Differential roles of ERK and p38 MAP kinase pathways in positive and negative selection of T lymphocytes. Immunity 9:565‐574.
   Weiss, L., Whitmarsh, A.J., Yang, D.D., Rincon, M., Davis, R.J., and Flavell, R.A. 2000. Regulation of c‐Jun NH2‐terminal kinase (Jnk) gene expression during T cell activation. J. Exp. Med 191:139–146.
   Whitmarsh, A.J. and Davis, R.J. 1998. Structural organization of MAP kinase signaling modules by scaffold proteins in yeast and mammals. Trends Biochem. Sci. 23:481‐485.
   Yang, D.D., Conze, D., Whitmarsh, A.J., Barrett, T., Davis, R.J., Rincon, M., and Flavell, R.A. 1998a. Differentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2. Immunity 9:575–585.
   Yang, D.D., Dong, C., Wysk, M., Whitmarsh, A.J., Davis, R.J., and Flavell, R.A. 1998b. Defective T cell differentiation in the absence of Jnk1. Science 282:2092–2095.
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