Bak BH3 peptides antagonize Bcl-x(L) function and induce apoptosis throughcytochrome c-independent activation of caspases

The Bcl-2 homology 3 (BH3) domain is crucial for the death-inducing and dim erization properties of pro-apoptotic members of the Bcl-2 protein family, including Bak, Bar, and Bad. Here we report that synthetic peptides corresp onding to the BH3 domain of Bak bind to Bcl-x(L), antagonize its anti-apopt otic function, and rapidly induce apoptosis when delivered into intact cell s via fusion to the Antennapedia homeoprotein internalization domain. Treat ment of HeLa cells with the Antennapedia-BH3 fusion peptide resulted in pep tide internalization and induction of apoptosis within 2-3 h, as indicated by caspase activation and subsequent poly(ADP-ribose) polymerase cleavage, as well as morphological characteristics of apoptosis, A point mutation wit hin the BH3 peptide that blocks its ability to bind to Bcl-x(L) abolished i ts apoptotic activity, suggesting that interaction of the BH3 peptide with Eel-a-related death suppressors, such as Bcl-x(L), may be critical for its activity in cells. While overexpression of Bcl-x(L) can block BH3-induced a poptosis, treatment with BH3 peptides resensitized Bcl-x(L)-expressing cell s to Fas-mediated apoptosis, BH3-induced apoptosis was blocked by caspase i nhibitors, demonstrating a dependence on caspase activation, but was not ac companied by a dramatic early loss of mitochondrial membrane potential or d etectable translocation of cytochrome c from mitochondria to cytosol, These findings demonstrate that the BH3 domain itself is capable of inducing apo ptosis in whole cells, possibly by antagonizing the function of Eel-a-relat ed death suppressors.