title: Rapid mutagenesis-based analysis of phosphorylation sites in mitogen-activated protein kinase substrates.
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.
authors.affiliation: Department of Stress & Developmental Biology, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120, Halle/Saale, Germany.
authors.forename: Lennart
authors.initials: L
authors.lastname: Eschen-Lippold
authors.forename: Nicole
authors.initials: N
authors.lastname: Bauer
authors.forename: Julia
authors.initials: J
authors.lastname: Löhr
authors.forename: Mieder A T
authors.initials: MA
authors.lastname: Palm-Forster
authors.forename: Justin
authors.initials: J
authors.lastname: Lee
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D013997:Time Factors; D014170:Transformation, Genetic
chemical_list: D017931:DNA Primers; D020928:Mitogen-Activated Protein Kinases