Rapid Mutagenesis-Based Analysis of Phosphorylation Sites in Mitogen-Activated Protein Kinase Substrates

  • Lennart Eschen-Lippold
  • Nicole Bauer
  • Julia Löhr
  • Mieder A. T. Palm-Forster
  • Justin LeeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1171)


In eukaryotes, mitogen-activated protein kinases (MAPKs) are one of the best studied pathways for posttranslational modification-mediated regulation of protein functions. Here, we describe a rapid in vitro method to screen potential protein phosphorylation sites targeted by MAPKs. The method is based on PCR-mediated mutagenesis together with a type IIs restriction digest. Screening for the successfully mutated clones is further facilitated through introduction of a second diagnostic restriction site. Besides time-saving, this reduces the cost for sequencing confirmation of the positive clones, which are used for subsequent recombinant protein production and kinase assay validation.

Key words

MAPK substrates Phosphorylation Mutagenesis 



M.A.T.P-.F. and L.E.-L. are supported by the Saxony-Anhalt Excellence Network Graduate program (W21040908) and the ProNET-T3 program (03ISO2211B), respectively. MAPK signaling work is financed by the Collaborative Research Center program, SFB648.


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Lennart Eschen-Lippold
    • 1
  • Nicole Bauer
    • 1
  • Julia Löhr
    • 1
  • Mieder A. T. Palm-Forster
    • 1
  • Justin Lee
    • 1
    Email author
  1. 1.Department of Stress & Developmental BiologyLeibniz Institute of Plant BiochemistryHalle/SaaleGermany

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