Cell Damage-Induced Conformational Changes of the Pro-Apoptotic Protein Bak in Vivo Precede the Onset of Apoptosis

• Published in: The Journal of cell biology Vol. 144; no. 5; pp. 903 - 914
• Main Authors: Griffiths, Gareth J., Dubrez, Laurence, Morgan, Clive P., Jones, Neil A., Whitehouse, Jenna, Corfe, Bernard M., Dive, Caroline, Hickman, John A.
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 08.03.1999; The Rockefeller University Press
• Subjects: Antibodies; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Caspases - metabolism; Cell Cycle; Cell lines; Cells; Cellular biology; Cellular immunity; Dexamethasone - pharmacology; Electrophoresis, Gel, Two-Dimensional; Enzyme Activation; Epitopes; Epitopes - chemistry; Etoposide - pharmacology; Fluorescence; Fluorescent antibody techniques; Histograms; Humans; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Microscopy, Fluorescence; Protein Conformation; Proteins; Regular; Staurosporine - pharmacology; Subcellular Fractions - metabolism; T lymphocytes; Tumor Cells, Cultured
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.144.5.903

Investigation of events committing cells to death revealed that a concealed NH2-terminal epitope of the pro-apoptotic protein Bak became exposed in vivo before apoptosis. This occurred after treatment of human Jurkat or CEM-C7A T-lymphoma cells with the mechanistically disparate agents staurosporine, etoposide or dexamethasone. The rapid, up to 10-fold increase in Bak-associated immunofluorescence was measured with epitope-specific monoclonal antibodies using flow cytometry and microscopy. In contrast, using a polyclonal antibody to Bak, immunofluorescence was detected both before and after treatment. There were no differences in Bak protein content nor in subcellular location before or after treatment. Immunofluorescence showed Bcl-xLand Bak were largely associated with mitochondria and in untreated cells they coimmunoprecipitated in the presence of nonioinic detergent. This association was significantly decreased after cell perturbation suggesting that Bcl-xLdissociation from Bak occurred on exposure of Bak's NH2terminus. Multiple forms of Bak protein were observed by two dimensional electrophoresis but these were unchanged by inducers of apoptosis. This indicated that integration of cellular damage signals did not take place directly on the Bak protein. Release of proteins, including Bcl-xL, from Bak is suggested to be an important event in commitment to death.