N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death.
Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.
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Drug Discovery Program, H. Lee Moffitt Cancer Center & Research Institute, and Department of Biochemistry & Molecular Biology, College of Medicine, University of South Florida, Tampa, Florida 33612, USA.
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D017209:Apoptosis; D001483:Base Sequence; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D017931:DNA Primers; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D019169:Jurkat Cells; D008928:Mitochondria / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D003574:Cytochrome c Group; D017931:DNA Primers; D004151:Dipeptides; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C071482:calpeptin; D005047:Etoposide; D002154:Calpain