Figure 3.

Determination of the caspase-3 cleavage sites in PKN. (A) Cleavage of PKN point mutants replacing Asp with Ala by caspase-3. Extracts of COS-7 cells expressing the FLAG-tagged PKN mutants were incubated with or without recombinant His-caspase-3 (Casp-3), and were analyzed for the cleavage of PKN by immunoblotting with PKN (C-19). Mutated amino acid number is shown on the top. White and black arrowheads on the right of the blot indicate the uncleaved and cleaved bands, respectively. Gray arrowhead indicates the cleavage product detected only in the in vitro reaction. rAF3 indicates the recombinant AF3-FLAG expressed in COS-7 cells. Molecular mass markers in kDa are indicated on the left. (B) Schematic representation of the structure of human PKN with the predicted cleavage sites and locations of AF1, AF3, and the in vitro 70-kDa product. The mutated Asp residues giving rise to the resistance to the cleavage are presented with neighboring amino acid sequences below the structure of PKN. The predicted positions of AF1, AF3, and the 70-kDa fragment are shown on the bottom. LZ, leucine zipper-like motif; BR, basic region.