Advertisement

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
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1171)

Abstract

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 

Notes

Acknowledgement

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.

References

  1. 1.
    Colcombet J, Hirt H (2008) Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem J 413:217–226PubMedCrossRefGoogle Scholar
  2. 2.
    Suarez Rodriguez MC, Petersen M, Mundy J (2010) Mitogen-activated protein kinase signaling in plants. Annu Rev Plant Biol 61:621–649CrossRefGoogle Scholar
  3. 3.
    Andreasson E, Ellis B (2010) Convergence and specificity in the Arabidopsis MAPK nexus. Trends Plant Sci 15:106–113PubMedCrossRefGoogle Scholar
  4. 4.
    Palm-Forster MAT, Eschen-Lippold L, Lee J (2012) A mutagenesis-based screen to rapidly identify phosphorylation sites in mitogen-activated protein kinase substrates. Anal Biochem 427:127–129PubMedCrossRefGoogle Scholar
  5. 5.
    Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300PubMedCentralPubMedGoogle Scholar
  6. 6.
    Dyson MR, Shadbolt SP, Vincent KJ, Perera RL, McCafferty J (2004) Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnol 4. doi: 10.1186/1472-6750-4-32
  7. 7.
    Lee J, Rudd JJ, Macioszek VK, Scheel D (2004) Dynamic changes in the localization of MAPK cascade components controlling pathogenesis-related (PR) gene expression during innate immunity in parsley. J Biol Chem 279:22440–22448PubMedCrossRefGoogle 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 throughput identification of potential Arabidopsis mitogen-activated protein kinase substrates. Mol Cell Proteomics 4:1558–1568PubMedCrossRefGoogle Scholar
  9. 9.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar

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

Personalised recommendations