MAP kinase ERK maintains specificity by binding to docking sites such as the DEF domain or D domain. It was previously shown that appending peptides derived from D domains to a substrate peptide increased apparent efficiency of peptide phosphorylation while preserving its apparent specificity for ERK. Here we determine the effect of the DEF motif on efficiency and specificity of peptide phosphorylation by ERK. The DEF motif modulated the apparent affinity of the peptide for ERK while the substrate motif dominated the apparent catalytic rate. Attachment of the DEF sequence improved apparent phosphorylation efficiency by 60-fold. Addition of peptides possessing both the DEF and D motif to a substrate sequence did not yield additive effects on the K-M of the substrate for ERK. Further, the DEF motif diminished the apparent specificity for ERK and increased the apparent efficiencies of phosphorylation of the substrate peptide by p38 alpha kinase and JNK1. (C) 2009 Elsevier Inc. All rights reserved.