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.
Wood
D E
DE
Department of Pathology, New York University Medical Center and Kaplan Comprehensive Cancer Center, New York 10016, USA.
Thomas
A
A
Devi
L A
LA
Berman
Y
Y
Beavis
R C
RC
Reed
J C
JC
Newcomb
E W
EW
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