Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells

Granzyme B (GrB), a serine protease with substrate specificity similar to t he caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apo ptosis predominantly via Bid cleavage, mitochondrial outer membrane permeab ilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicit y system, wherein target cells are treated with purified GrB and replicatio n-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic fam ily member, Bak, plays a dominant role in GrB-mediated mitochondrial apopto tic events. A variant of Jurkat cells, deficient in Bak expression, was res istant to GrB/Ad-mediated apoptosis, as determined by lack of membranous ph osphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytot oxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free s ystem using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release c ytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mi tochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to re port a role for Bak in GrB-mediated mitochondrial apoptosis. This study dem onstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and ther efore, suggests that deficiency in Bak may serve as a mechanism of immune e vasion for tumor or viral infected cells.