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Screening of Kinase Substrates Using Kinase Knockout Mutants

  • Taishi UmezawaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1306)

Abstract

Protein kinases are widely known to be major regulators of various signaling processes, particularly in eukaryotes, including plants. To understand their role in signal transduction pathways, it is necessary to determine which proteins are phosphorylated by these enzymes. Recent studies have applied a comparative phosphoproteomic approach to identify protein kinase substrates in plants. The results demonstrated that kinase knockout mutants are useful for screening protein kinase substrates via such a comparative analysis. Here some technical points are described for the experimental design and comparative analysis using kinase knockout mutants.

Key words

Knockout mutant Plant Phosphoproteomics Protein kinase Signal transduction 

Notes

Acknowledgements

This work was supported by the Japan Science and Technology Agency program PRESTO, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References

  1. 1.
    Lehti-Shiu MD, Shiu S-H (2012) Diversity, classification and function of the plant protein kinase superfamily. Philos Trans Roy Soc B Biol Sci 367:2619–2639CrossRefGoogle Scholar
  2. 2.
    Ishihama Y, Imami K (2014) Quantitation of cellular phosphorylation dynamics by phosphoproteomics approach. Yakugaku Zasshi 134:521–527CrossRefPubMedGoogle Scholar
  3. 3.
    Popescu SC et al (2009) MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays. Genes Dev 23:80–92CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Vlad F et al (2008) A versatile strategy to define the phosphorylation preferences of plant protein kinases and screen for putative substrates. Plant J 55:104–117CrossRefPubMedGoogle Scholar
  5. 5.
    Nakagami H et al (2012) Shotguns in the front line: phosphoproteomics in plants. Plant Cell Physiol 53:118–124CrossRefPubMedGoogle Scholar
  6. 6.
    Thelen JJ, Peck SC (2007) Quantitative proteomics in plants: choices in abundance. Plant Cell 19:3339–3346CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    De la Fuente van Bentem S, Hirt H (2007) Using phosphoproteomics to reveal signalling dynamics in plants. Trends Plant Sci 12:404–411CrossRefPubMedGoogle Scholar
  8. 8.
    Schreiber TB et al (2008) Quantitative phosphoproteomics - an emerging key technology in signal-transduction research. Proteomics 8:4416–4432CrossRefPubMedGoogle Scholar
  9. 9.
    Robitaille AM et al (2013) Quantitative phosphoproteomics reveal mTORC1 activates de novo pyrimidine synthesis. Science 339:1320–1323CrossRefPubMedGoogle Scholar
  10. 10.
    Kettenbach AN et al (2011) Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells. Sci Signal 4:rs5CrossRefPubMedGoogle Scholar
  11. 11.
    Koch A et al (2011) Mitotic substrates of the kinase Aurora with roles in chromatin regulation identified through quantitative phosphoproteomics of fission yeast. Sci Signal 4:rs6CrossRefPubMedGoogle Scholar
  12. 12.
    Umezawa T et al (2013) Genetics and phosphoproteomics reveal a protein phosphorylation network in the abscisic acid signaling pathway in Arabidopsis thaliana. Sci Signal 6:rs8CrossRefPubMedGoogle Scholar
  13. 13.
    Wang P et al (2013) Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action. Proc Natl Acad Sci 110:11205–11210CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Umezawa T et al (2009) Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis. Proc Natl Acad Sci U S A 106:17588–17593CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Sugiyama N et al (2007) Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications. Mol Cell Proteomics 6:1103–1109CrossRefPubMedGoogle Scholar
  16. 16.
    Chou MF, Schwartz D (2011) Biological sequence motif discovery using motif-x. Curr. Protoc. Bioinforma. Ed. Board Andreas Baxevanis Al Chapter 13, Unit 13.15–24Google Scholar
  17. 17.
    Nakagami H (2014) StageTip-based HAMMOC, an efficient and inexpensive phosphopeptide enrichment method for plant shotgun phosphoproteomics. In: Jorrin-Novo JV et al (eds) Plant proteomics: methods and protocols, vol 1072, Methods in molecular biology. Humana Press, Totowa, NJ, pp 595–607CrossRefGoogle Scholar
  18. 18.
    Sambrook J (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Faculty of AgricultureTokyo University of Agriculture and TechnologyKoganeiJapan
  2. 2.PRESTOJapan Science and Technology AgencySaitamaJapan

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