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Caspase cleavage enhances the apoptosis-inducing effects of BAD
F Condorelli1, P Salomoni, S Cotteret
1Department of Microbiology/Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Abstract:
The function of BAD, a proapoptotic member of the Bcl-2 family, is regulated primarily by rapid changes in phosphorylation that modulate its protein-protein interactions and subcellular localization. We show here that, during interleukin-3 (IL-3) deprivation-induced apoptosis of 32Dcl3 murine myeloid precursor cells, BAD is cleaved by a caspase(s) at its N terminus to generate a 15-kDa truncated protein. The 15-kDa truncated BAD is a more potent inducer of apoptosis than the wild-type protein, whereas a mutant BAD resistant to caspase 3 cleavage is a weak apoptosis inducer. Truncated BAD is detectable only in the mitochondrial fraction, interacts with BCL-X(L) at least as effectively as the wild-type protein, and is more potent than wild-type BAD in inducing cytochrome c release. Human BAD, which is 43 amino acids shorter than its mouse counterpart, is also cleaved by a caspase(s) upon exposure of Jurkat T cells to anti-FAS antibody, tumor necrosis factor alpha (TNF-alpha), or TRAIL. Moreover, a truncated form of human BAD lacking the N-terminal 28 amino acids is more potent than wild-type BAD in inducing apoptosis. The generation of truncated BAD was blocked by Bcl-2 in IL-3-deprived 32Dcl3 cells but not in Jurkat T cells exposed to anti-FAS antibody, TNF-alpha, or TRAIL. Together, these findings point to a novel and important role for BAD in maintaining the apoptotic phenotype in response to various apoptosis inducers.
Insights
Proapoptotic BAD protein is cleaved by caspases into a truncated form that enhances apoptosis. This truncated BAD, found at mitochondria, is a potent inducer of cell death, highlighting its role in apoptosis regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The proapoptotic protein BAD regulates cell death through phosphorylation, affecting its interactions and location.
- Understanding BAD's regulation is crucial for controlling apoptosis in various cellular contexts.
Purpose of the Study:
- To investigate the role of BAD cleavage by caspases in apoptosis.
- To characterize the function and localization of truncated BAD.
- To compare the apoptotic potency of truncated BAD with wild-type BAD.
Main Methods:
- Apoptosis induction in 32Dcl3 murine myeloid precursor cells and Jurkat T cells.
- Analysis of BAD cleavage by caspases using Western blotting and protein assays.
- Subcellular fractionation to determine the localization of truncated BAD.
- Assessment of protein-protein interactions with BCL-X(L).
- Measurement of cytochrome c release.
Main Results:
- BAD is cleaved by caspases at the N terminus during IL-3 deprivation-induced apoptosis, generating a 15-kDa truncated form.
- Truncated BAD is a more potent inducer of apoptosis than wild-type BAD.
- Truncated BAD localizes to the mitochondria and enhances cytochrome c release.
- Human BAD is also cleaved by caspases in response to various death signals, and its truncated form is more potent.
- Bcl-2 blocked truncated BAD generation in IL-3-deprived cells but not in Jurkat T cells.
Conclusions:
- Caspase-mediated cleavage generates a potent proapoptotic form of BAD.
- Truncated BAD plays a significant role in the apoptotic pathway, particularly at the mitochondria.
- BAD cleavage represents a novel mechanism contributing to the apoptotic phenotype.
- The regulation of BAD cleavage may differ depending on the apoptosis inducer and cell type.