FIG. 3.

Differential phosphorylation of MafA by members of the MAPK family in vitro. (A) HeLa cells were transfected with expression vectors for ERK2, p38α, JNK1, or ERK5 HA1-tagged MAPKs and stimulated, respectively, with TPA, UV, UV, or by cotransfection with an expression plasmid for MEK5DD, an activated version of MEK5. Kinases were immunoprecipitated with anti-HA1 antibody and incubated with [γ-³²P]ATP and GST-MafA recombinant protein, followed by Western blot and either direct autoradiography or incubation with anti-GST antibody. Positive controls (myelin basic protein [MBP] for ERK2, GST-ATF2 for p38α, GST-Jun for JNK1, and GST-MEF2C for ERK5) were treated likewise. (B) GST-MafA fusion proteins containing either truncated (left panels) or mutated (right panels) forms of MafA were subjected to kinase assay with recombinant activated ERK2. MafA, GST fused to full-length MafA; Nter, GST fused to the N-terminal half of MafA; Cter, GST fused to the C-terminal half of MafA; S14A, S65A, and S14A/S65A, GST fused to full-length MafA carrying the respective mutations. In vitro kinase assays were analyzed as described for panel A. (C) GST-MafA, either WT or carrying the S14A/S65A mutation, was subjected to in vitro kinase assay with activated p38α immunoprecipitated from HeLa cells as described for panel A.