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| Item Type: | Article |
|---|---|
| Title: | Association of active caspase 8 with the mitochondrial membrane during apoptosis: potential roles in cleaving BAP31 and caspase 3 and mediating mitochondrion-endoplasmic reticulum cross talk in etoposide-induced cell death |
| Creators Name: | Chandra, D. and Choy, G. and Deng, X. and Bhatia, B. and Daniel, P. and Tang, D.G. |
| Abstract: | It was recently demonstrated that during apoptosis, active caspase 9 and caspase 3 rapidly accumulate in the mitochondrion-enriched membrane fraction (D. Chandra and D. G. Tang, J. Biol. Chem.278:17408-17420, 2003). We now show that active caspase 8 also becomes associated with the membranes in apoptosis caused by multiple stimuli. In MDA-MB231 breast cancer cells treated with etoposide (VP16), active caspase 8 is detected only in the membrane fraction, which contains both mitochondria and endoplasmic reticulum (ER), as revealed by fractionation studies. Immunofluorescence microscopy, however, shows that procaspase 8 and active caspase 8 predominantly colocalize with the mitochondria. Biochemical analysis demonstrates that both procaspase 8 and active caspase 8 are localized mainly on the outer mitochondrial membrane (OMM) as integral proteins. Functional analyses with dominant-negative mutants, small interfering RNAs, peptide inhibitors, and Fas-associated death domain (FADD)- and caspase 8-deficient Jurkat T cells establish that the mitochondrion-localized active caspase 8 results mainly from the FADD-dependent and tumor necrosis factor receptor-associated death domain-dependent mechanisms and that caspase 8 activation plays a causal role in VP16-induced caspase 3 activation and cell death. Finally, we present evidence that the OMM-localized active caspase 8 can activate cytosolic caspase 3 and ER-localized BAP31. Cleavage of BAP31 leads to the generation of ER- localized, proapoptotic BAP20, which may mediate mitochondrion-ER cross talk through a Ca(2+)-dependent mechanism. |
| Keywords: | Apoptosis, Biological Models, Calcium, Caspase 3, Caspase 8, Caspases, Cell Death, Cell Line, Cell Membrane, Cell-Free System, Density Gradient Centrifugation, Down-Regulation, Drug Dose-Response Relationship, Endopeptidase K, Endoplasmic Reticulum, Enzyme Activation, Etoposide, Fluorescence Microscopy, Jurkat Cells, Membrane Proteins, Mitochondria, Phytogenic Antineoplastic Agents, Protein Binding, Salts, Small Interfering RNA, Subcellular Fractions, Sucrose, Tertiary Protein Structure, Transfection, Tumor Cell Line, Western Blotting |
| Source: | Molecular and Cellular Biology |
| ISSN: | 0270-7306 |
| Publisher: | American Society for Microbiology |
| Volume: | 24 |
| Page Range: | 6592-6607 |
| Date: | 1 January 2004 |
| Official Publication: | https://doi.org/10.1128/MCB.24.15.6592-6607.2004 |
| PubMed: | View item in PubMed |
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