FIG. 9.

Mapping of caspase 6 cleavage site by site-directed mutagenesis. (A) Nucleotide and amino acid sequences of wild-type (WT) AP-2α and mutant AP-2 M1-3. A downward arrow after D¹⁹ shows the caspase 6 cleavage site of WT AP-2α. A single base substitution of C for A in the GAC codon (underlined) mutated D¹⁹ to A¹⁹. (B) Control WT IVT AP-2α (lanes 1 and 7) and control mutant IVT AP-2 M1-3 (lanes 3 and 10). Using 12.5 U per reaction, caspases 1 and 3 did not cleave WT IVT AP-2α (lanes 2 and 3) or mutant AP-2 M1-3 (lanes 5 and 6). Caspase 6 cleaved only WT AP-2α at 1 U per reaction (lane 8), and a caspase 6-preferred inhibitor abrogated this cleavage (lane 9). In contrast, caspase 6 did not cleave mutant AP-2 M1-3 (lane 11), indicating that D¹⁹ is the cleavage site for caspase 6.