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