Figure 3

Mutation of key residues diminishes EAS-3 phosphorylation by pERK2. (A) Primary sequences of EAS-3 and EAS-3 mutant linker peptides are shown with consensus phosphorylation sites (highlighted in green) and ERK binding sites (underlined). Residue mutations to alanine are highlighted in red. (B) Analysis of mutant EAS-3 proteins by γ-[³²P]ATP phosphorylation shows complete loss of pERK2 phosphorylation of the S(286,292)A mutant as compared to wild-type EAS-3. Decreased ³²P-phosphorylation of site-specific mutants also indicates the involvement of both Ser286 and Ser292 for pERK2 activity. Requirement of the ERK binding domain for efficient phosphorylation is evident from decreased phosphorylation of the EAS3-DBD mutant. Band intensities were quantitated by densitometry and are shown graphically. Equal protein loading is shown by Coomassie staining of EAS-3 and EAS-3 mutants and phosphorylation assay was terminated after 15 minutes at 30°C. (C) Fluorimetry data for EAS-3 and EAS-3 mutants reveals that the FRET efficiency change is reduced with various mutations, consistent with the phosphorylation assay in B.