Caspase-3-dependent Cleavage of Bcl-2 Promotes Release of Cytochrome c

doi:10.1074/jbc.274.30.21155

Caspase-3-dependent Cleavage of Bcl-2 Promotes Release of Cytochrome c*

From the ^‡Oncology Center and the ^¶Departments of Molecular Microbiology and Immunology, Pharmacology and Molecular Sciences, and Neurology, Johns Hopkins Schools of Public Health and Medicine, Baltimore, Maryland 21205, ^§Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, and ^**National Institute of Aging, National Institutes of Health, Baltimore, Maryland 21224

Abstract

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.

Footnotes

↵* This work was supported by National Institutes of Health Grants GM07309 (to D. G. K.), ES05777 and CA77715 (to M. B. K.), CA13106–25 (to Y. A. L.), and NS34175 (to J. M. H.) and by a grant from the Muscular Dystrophy Association (to J. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ Present address: Dept. of Hematology-Oncology, St. Jude Children’s Research Hospital, Memphis, TN 38105.
↵‡ The Steven Birnbaum Scholar of The Leukemia Society of America.
↵§§ A Pew scholar.
↵¶¶ To whom correspondence should be addressed. Tel.: 410-955-2716; Fax: 410-955-0105; E-mail: hardwick@jhu.edu.
Abbreviations:

PARP

poly(ADP-ribose) polymerase

DEVD-CHO

N-acetyl-Asp-Glu-Val-aspartinal

YVAD-CHO

N-acetyl-Tyr-Val-Ala-aspartinal

PAGE

polyacrylamide gel electrophoresis

HA

hemagglutinin

BHK

baby hamster kidney

Z-VAD-fmk

N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone
- Received May 6, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.