Caspase-mediated cleavage of MRCKα at Asp 478 determines increase of its kinase activity. (A) Caspase-mediated MRCKα and MRCKβ cleavage assay in HeLa cells treated with 10 µM doxorubicin for 16 h in combination with increasing concentrations of the caspase inhibitor zVAD-FMK shows that MRCKα is cleaved in two separates sites and MRCKβ in only one site. Left, representative experiment. Right, quantification and mean of six experiments. Error bars represent SD. (B) MRCKα and MRCKβ primary structures showing the different domains (kinase domain, KD; coiled coils domains, CC; protein kinase C conserved region 1, C1; pleckstrin homology-like, PH; citron homology, CH; CDC42/Rac interactive binding, C), the two antibody epitopes, and the estimation of cleavage regions as derived from electrophoretic path length of the cleavage products. (C) Sequence of MRCKα including Asp 478 compared with the homologue sequence in MRCKβ. Yellow background, conserved amino acids; green background, amino acids with similar features. (D) Cleavage screening of putative cleavage sequences in sites 1 and 2 of MRCKα. As negative controls, all the aspartates have been mutated in alanines and compared in the same assay. Immunoblot with anti-GST and anti-GFP antibodies was used to detect the full-length (FL) proteins and their relative cleavage products (CPs). This experiment proves that the two most efficient cleavage sites of MRCKα are Asp 478 and Asp 971. (E) The increase of MRCKα kinase activity after caspase-mediated cleavage is shown by in vitro kinase assay. Pulled-down full-length MRCKα (wild type or D478A mutant) fused to GST were first incubated with recombinant-active caspase 3 then with an MRCKα-specific substrate. Error bars represent the SEM. Western blots show the efficiency of cleavage mediated by the recombinant-active caspase 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001.