Cellular damage signals promote sequential changes at the N-terminus and BH-1 domain of the pro-apoptotic protein Bak

The pro-apoptotic protein Bak is converted from a latent to an active form by damage-induced signals. This process involves an early exposure of an oc cluded N-terminal epitope of Bak in intact cells. Here we report a subseque nt damage-induced change in Bak, detected using an antibody to the central BH-1 domain. Bak coimmunoprecipitated with Bcl-x(L) both in undamaged cells and early after damage, when the N-terminal epitope was exposed but the BH -1 epitope remained occluded. A subsequent decrease in binding of Bak to Bc l-x(L) correlated with exposure of an epitope in the Bak BH-1 domain. Overe xpression of Bcl-x(L) did not affect the kinetics of exposure of the Bak N- terminal epitope but delayed exposure of the BH-1 domain. Cytochrome c rele ase from mitochondria facilitates the activation of apoptotic caspases. The majority of cells with exposed Bak BH-1 domains contained cytosolic cytoch rome c. However, a small proportion of cells exhibited exposed Bak BH-1 dom ains that co-localized with mitochondrial cytochrome c. The data are consis tent with a two-step model for the activation of Bak by drug-induced damage signals where dissociation of Bcl-x(L) from the BH-1 domain of Bak occurs immediately prior to or concomitantly with cytochrome c release.