A mutagenesis-based screen to rapidly identify phosphorylation sites in mitogen-activated protein kinase substrates

Identification and characterization of protein phosphorylation sites often requires mass spectrometric analysis, which is not trivial or accessible to many laboratories. Here, a targeted strategy to mutagenize putative phosphorylation sites within mitogen-activated protein kinase (MAPK) substrates is described. This employs a combination of standard type II with type IIs restriction enzymes to rapidly create individual or multiple phosphorylation site mutant versions of kinase substrates with high efficiency, thereby reducing the cost for screening mutated clones.