Figure 4

Determination of ERK2 specificity for EAS-3. (A) Phosphorylation of EAS-3 is efficient with pERK2, but not pp38 or pSAPK. pp38 and pSAPK incorporates little or no ³²P into EAS-3 at the same relative specific activity as pERK2. Band intensities were quantitated by densitometry and are shown graphically. Equal protein loading of EAS-3 is shown by Coomassie staining. (B) Specific targets for pERK2 (MBP), pp38 (GST-ATF2), and pSAPK (GST-cJun) are efficiently phosphorylated in the presence of γ-[³²P]ATP. The amounts of active kinase used in A were based on the relative incorporation of ³²P into each cognate substrate by the respective kinase. (C) Fluorimetry of EAS-3 with MAPKs shows that the FRET efficiency change is significantly diminished in the presence of pp38 or pSAPK as compared to pERK2. This is consistent with the phosphorylation assay in A.