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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.

Pubmed ID: 15254227


  • Chandra D
  • Choy G
  • Deng X
  • Bhatia B
  • Daniel P
  • Tang DG


Molecular and cellular biology

Publication Data

August 15, 2004

Associated Grants

  • Agency: NCI NIH HHS, Id: CA 90297
  • Agency: NIEHS NIH HHS, Id: ES 07784

Mesh Terms

  • Antineoplastic Agents, Phytogenic
  • Apoptosis
  • Blotting, Western
  • Calcium
  • Caspase 3
  • Caspase 8
  • Caspases
  • Cell Death
  • Cell Line
  • Cell Line, Tumor
  • Cell Membrane
  • Cell-Free System
  • Centrifugation, Density Gradient
  • Dose-Response Relationship, Drug
  • Down-Regulation
  • Endopeptidase K
  • Endoplasmic Reticulum
  • Enzyme Activation
  • Etoposide
  • Humans
  • Jurkat Cells
  • Membrane Proteins
  • Microscopy, Fluorescence
  • Mitochondria
  • Models, Biological
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA, Small Interfering
  • Salts
  • Subcellular Fractions
  • Sucrose
  • Transfection