Fig. 6. Dual phosphorylation on threonine and tyrosine is required for p38 MAPK activation. Epitope-tagged wild-type and mutated p38α (A and B) or p38β2 (C and D) were expressed in COS7 cells. The mutated p38 contained point mutations in the Thr-Gly-Tyr (TGY) dual phosphorylation motif (replacement of threonine and tyrosine with alanine and phenylalanine, respectively). The effect of treatment with 80 J/m² UV (A and C) or co-expression with activated MKK6 (B and D) was examined. Immune complex kinase assays were performed to measure p38 activity using ATF2 as the substrate. The expression of p38 and activated MKK6 was examined by immunoblot analysis (lower panel).