Caspases play important roles in regulating apoptotic signaling pathways. Here we report the cloning, by the yeast two hybrid system with dominant negative caspase-2 as "bait," of a proapoptotic molecule named proapoptotic caspase adaptor protein (PACAP), encoded by a 372-base pair open reading frame. Binding of this novel protein to caspase-2 (casp-2) was confirmed in yeast two hybrid, in vitro, and in vivo assays. The deduced amino acid sequence revealed homology to functional motifs, including ATP and cytochrome c binding sites. PACAP mRNA was widely expressed in most human tissues; in transfected cells, PACAP was diffusely expressed in the cytoplasm. Bindings studies with the PACAP recombinant protein demonstrated specific binding to casp-2 and casp-9 but not to casp-3, -4, -7, or -8 in cell extracts. Cotransfection experiments showed that PACAP binds to casp-2 and -9 in 293T cells. In addition, studies with truncated PACAP demonstrated a requirement for residues 39-72 of PACAP for specific binding to casp-2 and -9. Transient transfection of PACAP into 293T human kidney cells and rat-1 fibroblasts triggered apoptosis at 24 h, which was at least in part prevented by an inhibitor of casp-3-like enzymes. Transfection of PACAP into human B cell lines using a retroviral system also triggered apoptotic cell death. In addition, transcription of PACAP in primary human B cells was dramatically down-regulated early after cellular activation by CD40L and Staphylococcus aureus and markedly up-regulated as the cells apoptose. These findings identify a novel proapoptotic caspase adaptor protein.