Identification of MAPK Substrates Using Quantitative Phosphoproteomics

  • Tong Zhang
  • Jacqueline D. Schneider
  • Ning Zhu
  • Sixue ChenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)


Activation of mitogen-activated protein kinases (MAPKs) under diverse biotic and abiotic factors and identification of an array of downstream MAPK target proteins are hot topics in plant signal transduction. Through interactions with a plethora of substrate proteins, MAPK cascades regulate many physiological processes in the course of plant growth, development, and response to environmental factors. Identification and quantification of potential MAPK substrates are essential, but have been technically challenging. With the recent advancement in phosphoproteomics, here we describe a method that couples metal dioxide for phosphopeptide enrichment with tandem mass tags (TMT) mass spectrometry (MS) for large-scale MAPK substrate identification and quantification. We have applied this method to a transient expression system carrying a wild type (WT) and a constitutive active (CA) version of a MAPK. This combination of genetically engineered MAPKs and phosphoproteomics provides a high-throughput, unbiased analysis of MAPK-triggered phosphorylation changes on the proteome scale. Therefore, it is a robust method for identifying potential MAPK substrates and should be applicable in the study of other kinase cascades in plants as well as in other organisms.

Key words

MAPK MAPK substrate Signal transduction Phosphoproteomics TMT 



This research was supported by grants from the National Science Foundation (MCB 0818051 and MCB 1412547) to S. Chen.


  1. 1.
    Pitzschke A (2015) Modes of MAPK substrate recognition and control. Trends Plant Sci 20:49–55CrossRefPubMedGoogle Scholar
  2. 2.
    Chinchilla D, Zipfel C, Robatzek S, Kemmerling B, Nurnberger T, Jones JDG et al (2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448:497–500CrossRefPubMedGoogle Scholar
  3. 3.
    Lu DP, Lin WW, Gao XQ, Wu SJ, Cheng C, Avila J et al (2011) Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity. Science 332:1439–1442CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Meng XZ, Wang HC, He YX, Liu YD, Walker JC, Torii KU et al (2012) A MAPK cascade downstream of ERECTA receptor-like protein kinase regulates Arabidopsis inflorescence architecture by promoting localized cell proliferation. Plant Cell 24:4948–4960CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Yamaguchi K, Yamada K, Ishikawa K, Yoshimura S, Hayashi N, Uchihashi K et al (2013) A receptor-like cytoplasmic kinase targeted by a plant pathogen effector Is directly phosphorylated by the chitin receptor and mediates rice immunity. Cell Host Microbe 13:347–357CrossRefPubMedGoogle Scholar
  6. 6.
    Mao GH, Meng XZ, Liu YD, Zheng ZY, Chen ZX, Zhang SQ (2011) Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell 23:1639–1653CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Andreasson E, Jenkins T, Brodersen P, Thorgrimsen S, Petersen NHT, Zhu SJ et al (2005) The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J 24:2579–2589CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Xu J, Chua NH (2012) Dehydration stress activates Arabidopsis MPK6 to signal DCP1 phosphorylation. EMBO J 31:1975–1984CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Feilner T, Hultschig C, Lee J, Meyer S, Immink RGH, Koenig A et al (2005) High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates. Mol Cell Proteomics 4:1558–1568CrossRefPubMedGoogle Scholar
  10. 10.
    Minkoff BB, Stecker KE, Sussman MR (2015) Rapid phosphoproteomic effects of ABA on wildtype and ABA receptor-deficient A. thaliana mutants. Mol Cell Proteomics 14:1169–1182CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Wang P, Xue L, Batelli G, Lee S, Hou YJ, Van Oosten MJ et al (2013) Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action. Proc Natl Acad Sci USA 110:11205–11210CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kline-Jonakin KG, Barrett-Wilt GA, Sussman MR (2011) Quantitative plant phosphoproteomics. Curr Opin Plant Biol 14:507–511CrossRefPubMedGoogle Scholar
  13. 13.
    Reiland S, Finazzi G, Endler A, Willig A, Baerenfaller K, Grossmann J et al (2011) Comparative phosphoproteome profiling reveals a function of the STN8 kinase in fine-tuning of cyclic electron flow (CEF). Proc Natl Acad Sci USA 108:12955–12960CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Hoehenwarter W, Thomas M, Nukarinen E, Egelhofer V, Rohrig H, Weckwerth W et al (2013) Identification of novel in vivo MAP kinase substrates in Arabidopsis thaliana through use of tandem metal oxide affinity chromatography. Mol Cell Proteomics 12:369–380CrossRefPubMedGoogle Scholar
  15. 15.
    Cheng Z, Li JF, Niu Y, Zhang XC, Woody OZ, Xiong Y et al (2015) Pathogen-secreted proteases activate a novel plant immune pathway. Nature 521:213–216CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Berriri S, Garcia AV, Frey NFD, Rozhon W, Pateyron S, Leonhardt N et al (2012) Constitutively active mitogen-activated protein kinase versions reveal functions of Arabidopsis MPK4 in pathogen defense Signaling. Plant Cell 24:4281–4293CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Li JN, Silva-Sanchez C, Zhang T, Chen SX, Li HY (2015) Phosphoproteomics technologies and applications in plant biology research. Front Plant Sci 6:430PubMedPubMedCentralGoogle Scholar
  18. 18.
    Zhang T, Zhu MM, Song WY, Harmon AC, Chen SX (2015) Oxidation and phosphorylation of MAP kinase 4 cause protein aggregation. Biochim Biophys Acta (BBA-Proteins Proteom) 1854:156–165CrossRefGoogle Scholar
  19. 19.
    Chen Y, Britton DJ, Paraiso K, Fedorenko I, Wood ER, Magliocco A et al (2014) Quantification of biomarker expression, phosphorylation, and mutation in cancer using TMT labeling prior to liquid chromatography-multiple reaction monitoring mass spectrometry. Cancer Res 74:2491Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Tong Zhang
    • 1
    • 2
  • Jacqueline D. Schneider
    • 1
    • 3
  • Ning Zhu
    • 1
  • Sixue Chen
    • 1
    • 2
    • 4
    • 5
    Email author
  1. 1.Department of BiologyUniversity of FloridaGainesvilleUSA
  2. 2.Genetics InstituteUniversity of FloridaGainesvilleUSA
  3. 3.Department of Chemical EngineeringUniversity of FloridaGainesvilleUSA
  4. 4.Plant Molecular and Cellular Biology ProgramUniversity of FloridaGainesvilleUSA
  5. 5.Interdisciplinary Center for Biotechnology ResearchUniversity of FloridaGainesvilleUSA

Personalised recommendations