Analysis of MAPK Activities Using MAPK-Specific Antibodies

  • Roland Willmann
  • Daniel J. Haischer
  • Andrea A. GustEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1171)


Phosphorylation of proteins by mitogen-activated protein kinases is central to many cellular processes, including signal transduction after stress encounter. Thus, assays to identify or characterize MAP kinase activities are a key tool for research in this area. While in-gel kinase assays using isotope-labeled ATP are a powerful tool to investigate the general induction of MAPK activities in any organism, alternative methods using phospho-specific MAPK antibodies are now being established for many model organisms. However, both in-gel kinase assay and phospho-specific western blot analysis do not allow for the unambiguous identification of the activated MAPK. To obtain specificity, initial immunoprecipitation purification of the kinase of interest prior to further analysis can be performed.

Key words

AtMPK3 AtMPK4 AtMPK6 Immunocomplex kinase assay Phospho-specific MAPK antibody Western blot 



We thank the Deutsche Forschungsgemeinschaft (SFB 766 and BR3875/1-1 as part of the ERA-PG PRR CROP consortium) for support to R.W. and A.A.G.


  1. 1.
    Rodriguez MC, Petersen M, Mundy J (2010) Mitogen-activated protein kinase signaling in plants. Annu Rev Plant Biol 61:621–649PubMedCrossRefGoogle Scholar
  2. 2.
    Ligterink W (2000) MAP kinases in plant signal transduction: how many, and what for? Results Probl Cell Differ 27:11–27PubMedCrossRefGoogle Scholar
  3. 3.
    Mishra NS, Tuteja R, Tuteja N (2006) Signaling through MAP kinase networks in plants. Arch Biochem Biophys 452:55–68PubMedCrossRefGoogle Scholar
  4. 4.
    Jonak C, Okresz L, Bogre L, Hirt H (2002) Complexity, cross talk and integration of plant MAP kinase signalling. Curr Opin Plant Biol 5:415–424PubMedCrossRefGoogle Scholar
  5. 5.
    Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415:977–983PubMedCrossRefGoogle Scholar
  6. 6.
    Pedley KF, Martin GB (2005) Role of mitogen-activated protein kinases in plant immunity. Curr Opin Plant Biol 8:541–547PubMedCrossRefGoogle Scholar
  7. 7.
    Colcombet J, Hirt H (2008) Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem J 413:217–226PubMedCrossRefGoogle Scholar
  8. 8.
    Feilner T, Hultschig C, Lee J, Meyer S, Immink RG, Koenig A, Possling A, Seitz H, Beveridge A, Scheel D, Cahill DJ, Lehrach H, Kreutzberger J, Kersten B (2005) High through-put identification of potential Arabidopsis mitogen-activated protein kinases substrates. Mol Cell Proteomics 4:1558–1568Google Scholar
  9. 9.
    Sörensson C, Lenman M, Schopper S, Veide Vilg J, Ljungdahl T, Grotli M, Tamas MJ, Peck SC, Andreasson E (2012) Determination of primary sequence specificity of Arabidopsis MAPKs MPK3 and MPK6 leads to identification of new substrates. Biochem J 446:271–278PubMedCrossRefGoogle Scholar
  10. 10.
    Whitmarsh AJ (2007) Regulation of gene transcription by mitogen-activated protein kinase signaling pathways. Biochim Biophys Acta 1773:1285–1298PubMedCrossRefGoogle Scholar
  11. 11.
    Wooten MW (2002) In-gel kinase assay as a method to identify kinase substrates. Sci STKE 2002:pl15PubMedGoogle Scholar
  12. 12.
    Ludwig AA, Saitoh H, Felix G, Freymark G, Miersch O, Wasternack C, Boller T, Jones JD, Romeis T (2005) Ethylene-mediated cross-talk between calcium-dependent protein kinase and MAPK signaling controls stress responses in plants. Proc Natl Acad Sci U S A 102:10736–10741PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Romeis T, Piedras P, Zhang S, Klessig DF, Hirt H, Jones JD (1999) Rapid Avr9- and Cf-9-dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor, wound, and salicylate responses. Plant Cell 11:273–287PubMedCentralPubMedGoogle Scholar
  14. 14.
    Pedley KF, Martin GB (2004) Identification of MAPKs and their possible MAPK kinase activators involved in the Pto-mediated defense response of tomato. J Biol Chem 279:49229–49235PubMedCrossRefGoogle Scholar
  15. 15.
    Kishi-Kaboshi M, Okada K, Kurimoto L, Murakami S, Umezawa T, Shibuya N, Yamane H, Miyao A, Takatsuji H, Takahashi A, Hirochika H (2010) A rice fungal MAMP-responsive MAPK cascade regulates metabolic flow to antimicrobial metabolite synthesis. Plant J 63:599–612PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Ahlfors R, Macioszek V, Rudd J, Brosche M, Schlichting R, Scheel D, Kangasjarvi J (2004) Stress hormone-independent activation and nuclear translocation of mitogen-activated protein kinases in Arabidopsis thaliana during ozone exposure. Plant J 40:512–522Google Scholar
  17. 17.
    Anderson JC, Bartels S, Gonzalez Besteiro MA, Shahollari B, Ulm R, Peck SC (2011) Arabidopsis MAP kinase phosphatase 1 (AtMKP1) negatively regulates MPK6-mediated PAMP responses and resistance against bacteria. Plant J 67:258–268PubMedCrossRefGoogle Scholar
  18. 18.
    Beckers GJ, Jaskiewicz M, Liu Y, Underwood WR, He SY, Zhang S, Conrath U (2009) Mitogen-activated protein kinases 3 and 6 are required for full priming of stress responses in Arabidopsis thaliana. Plant Cell 21:944–953Google Scholar
  19. 19.
    Heese A, Hann DR, Gimenez-Ibanez S, Jones AM, He K, Li J, Schroeder JI, Peck SC, Rathjen JP (2007) The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc Natl Acad Sci U S A 104:12217–12222PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Samuel MA, Hall H, Krzymowska M, Drzewiecka K, Hennig J, Ellis BE (2005) SIPK signaling controls multiple components of harpin-induced cell death in tobacco. Plant J 42:406–416PubMedCrossRefGoogle Scholar
  21. 21.
    Segonzac C, Feike D, Gimenez-Ibanez S, Hann DR, Zipfel C, Rathjen JP (2011) Hierarchy and roles of pathogen-associated molecular pattern-induced responses in Nicotiana benthamiana. Plant Physiol 156:687–699PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Brock AK, Willmann R, Kolb D, Grefen L, Lajunen HM, Bethke G, Lee J, Nurnberger T, Gust AA (2010) The Arabidopsis mitogen-activated protein kinase phosphatase PP2C5 affects seed germination, stomatal aperture, and abscisic acid-inducible gene expression. Plant Physiol 153:1098–1111PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Baccarini M (2005) Second nature: biological functions of the Raf-1 “kinase”. FEBS Lett 579:3271–3277PubMedCrossRefGoogle Scholar
  24. 24.
    Meloche S, Pouyssegur J (2007) The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition. Oncogene 26:3227–3239PubMedCrossRefGoogle Scholar
  25. 25.
    Roux PP, Blenis J (2004) ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68:320–344PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Roberts PJ, Der CJ (2007) Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 26:3291–3310PubMedCrossRefGoogle Scholar
  27. 27.
    Bethke G, Pecher P, Eschen-Lippold L, Tsuda K, Katagiri F, Glazebrook J, Scheel D, Lee J (2012) Activation of the Arabidopsis thaliana mitogen-activated protein kinase MPK11 by the flagellin-derived elicitor peptide, flg22. Mol Plant Microbe Interact 25:471–480Google Scholar
  28. 28.
    Reuter CW, Catling AD, Weber MJ (1995) Immune complex kinase assays for mitogen-activated protein kinase and MEK. Methods Enzymol 255:245–256PubMedCrossRefGoogle Scholar
  29. 29.
    Bethke G, Unthan T, Uhrig JF, Poschl Y, Gust AA, Scheel D, Lee J (2009) Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Proc Natl Acad Sci U S A 106:8067–8072Google Scholar
  30. 30.
    Xie G, Kato H, Imai R (2012) Biochemical identification of the OsMKK6-OsMPK3 signalling pathway for chilling stress tolerance in rice. Biochem J 443:95–102PubMedCrossRefGoogle Scholar
  31. 31.
    Holley SR, Yalamanchili RD, Moura DS, Ryan CA, Stratmann JW (2003) Convergence of signaling pathways induced by systemin, oligosaccharide elicitors, and ultraviolet-B radiation at the level of mitogen-activated protein kinases in Lycopersicon peruvianum suspension-cultured cells. Plant Physiol 132:1728–1738PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Stulemeijer IJ, Stratmann JW, Joosten MH (2007) Tomato mitogen-activated protein kinases LeMPK1, LeMPK2, and LeMPK3 are activated during the Cf-4/Avr4-induced hypersensitive response and have distinct phosphorylation specificities. Plant Physiol 144:1481–1494PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Roland Willmann
    • 1
  • Daniel J. Haischer
    • 1
  • Andrea A. Gust
    • 1
    Email author
  1. 1.Department of Plant BiochemistryZMBP, University of TübingenTübingenGermany

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