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 Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax 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 Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax 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.
Department of Pathology, New York University Medical Center and Kaplan Comprehensive Cancer Center, New York 10016, USA.
D E
DE
Wood
A
A
Thomas
L A
LA
Devi
Y
Y
Berman
R C
RC
Beavis
J C
JC
Reed
E W
EW
Newcomb
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects*; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D002154:Calpain / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D002166:Camptothecin / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002457:Cell Extracts / Q000737:chemistry; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology; D018922:HL-60 Cells / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000187:drug effects / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D013379:Substrate Specificity; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D002457:Cell Extracts; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C071482:calpeptin; D001224:Aspartic Acid; C080125:9-aminocamptothecin; D011065:Poly(ADP-ribose) Polymerases; D002154:Calpain; D007930:Leucine; D000409:Alanine; C024974:E 64; D002166:Camptothecin