Several inhibitor of apoptosis proteins (IAPs) are cleaved during apoptosis. Studies of the melanoma-associated IAP (ML-IAP) Livin, using recombinant molecules, have implicated both caspases 3/7 and the serine protease Omi/HtrA2 in its proteolytic cleavage.
To characterize the apoptotic cleavage of Livin in melanocytic cells, and evaluate the role of known proteases.
We assessed the capacity of a variety of stimuli to induce Livin cleavage in human melanoma cell lines and normal human melanocytes. The role of caspases and Omi was examined using caspase inhibitors and RNAi, respectively. A potential caspase substrate was further examined by site-directed mutagenesis. Deletion mapping was used to identify the cleavage site.
Livin cleavage was observed in multiple human melanoma cell lines in response to a variety of apoptotic stimuli (UVB, 4-TBP, cisplatin, TNF, Bax), and not affected by the addition of various protease inhibitors or RNAi-mediated silencing of Omi/HtrA2. Livin cleavage induced by 4-TBP, but not UVB or cisplatin, was blocked by the pan-caspase inhibitor zVAD-fmk. Mutation of Asp52 to Glu in Livin did not affect cleavage, while either mutation of Asp52 to Ala, deletion of Asp52, or deletion of the adjacent region (residues 53–61) abrogated cleavage.
Livin cleavage, induced by multiple apoptotic stimuli in melanoma cells, likely occurs in an Omi-independent fashion at residue 52 within its potential caspase substrate (DHVD52). However, relative insensitivity of the apoptotic cleavage to zVAD-fmk, or Asp52 to Glu mutation, suggests the involvement of a non-canonical caspase.