Cell damage-induced conformational changes of the pro-apoptotic protein bak in vivo precede the onset of apoptosis

Investigation of events committing cells to death revealed that a concealed NH2-terminal epitope of the pro-apoptotic protein Bak became exposed in vi vo before apoptosis. This occurred after treatment of human Jurkat or CEM-C 7A T-lymphoma cells with the mechanistically disparate agents staurosporine , etoposide or dexamethasone. The rapid, up to 10-fold increase in Bak-asso ciated immunofluorescence was measured with epitope-specific monoclonal ant ibodies using flow cytometry and microscopy. In contrast, using a polyclona l antibody to Bak, immunofluorescence was detected both before and after tr eatment. There were no differences in Bak protein content nor in subcellula r location before or after treatment. Immunofluorescence showed Bcl-x(L) an d Bak were largely associated with mitochondria and in untreated cells they coimmunoprecipitated in the presence of nonioinic detergent. This associat ion was significantly decreased after cell perturbation suggesting that Bcl -x(L) dissociation from Bak occurred on exposure of Bak's NH2 terminus. Mul tiple forms of Bak protein were observed by two dimensional electrophoresis but these were unchanged by inducers of apoptosis, This indicated that int egration of cellular damage signals did not take place directly on the Bah protein. Release of proteins, including Bcl-x(L), from Bah is suggested to be an important event in commitment to death.