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.
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.
Chandra
Dhyan
D
Dept. of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Park Rd. 1C, Smithville, TX 78957, USA.
Choy
Grace
G
Deng
Xiaodi
X
Bhatia
Bobby
B
Daniel
Peter
P
Tang
Dean G
DG
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D019286:Endopeptidase K / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D014162:Transfection
D000972:Antineoplastic Agents, Phytogenic; C091247:BCAP31 protein, human; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D012492:Salts; D013395:Sucrose; D005047:Etoposide; D019286:Endopeptidase K; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases; D002118:Calcium