Rapid mutagenesis-based analysis of phosphorylation sites in mitogen-activated protein kinase substrates

Methods Mol Biol. 2014;1171:183-92. doi: 10.1007/978-1-4939-0922-3_15.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / enzymology
  • Arabidopsis / genetics
  • Base Sequence
  • Binding Sites
  • DNA Primers / genetics
  • Escherichia coli / genetics
  • Mitogen-Activated Protein Kinases / genetics
  • Mitogen-Activated Protein Kinases / isolation & purification
  • Mitogen-Activated Protein Kinases / metabolism*
  • Mutagenesis*
  • Mutation
  • Phosphorylation
  • Polymerase Chain Reaction
  • Protein Binding
  • Protein Engineering / methods*
  • Protein Processing, Post-Translational
  • Substrate Specificity
  • Time Factors
  • Transformation, Genetic


  • DNA Primers
  • Mitogen-Activated Protein Kinases