Bax cleavage is mediated by calpain during drug-induced apoptosis

The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Pax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis, Here me identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Pax which is absent from the cytosolic fraction, Pax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis, Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain, Partial purification of the Pax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Pax cleavage, In addition, purified calpain enzymes cleaved Pax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Pax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.