Characterization of a novel proapoptotic caspase-2-and caspase-9-binding protein

Caspases play important roles in regulating apoptotic signaling pathways. H ere we report the cloning, by the yeast two hybrid system with dominant neg ative caspase-2 as "bait," of a proapoptotic molecule named proapoptotic ca spase adaptor protein (PACA-P), encoded by a 372-base pair open reading fra me. Binding of this novel protein to caspase-2 (casp-2) was confirmed in ye ast two hybrid, in vitro, and in vivo assays. The deduced amino acid sequen ce revealed homology to functional motifs, including ATP and cytochrome c b inding sites. PACAP mRNA was widely expressed in most human tissues; in tra nsfected cells, PACAP was diffusely expressed in the cytoplasm. Bindings st udies with the PACAP recombinant protein demonstrated specific binding to c asp-2 and casp-9 but not to casp-3, -4, -7, or -8 in cell extracts. Cotrans fection experiments showed that PACAP binds to casp-2 and -9 in 293T cells. In addition, studies with truncated PACAP demonstrated a requirement for r esidues 39-72 of PACAP for specific binding to casp-2 and -9. Transient tra nsfection of PACAP into 293T human kidney cells and rat-1 fibroblasts trigg ered apoptosis at 24 h, which was at least in part prevented by an inhibito r of casp-3-like enzymes. Transfection of PACAP into human B cell lines usi ng a retroviral system also triggered apoptotic cell death. In addition, tr anscription of PACAP in primary human B cells was dramatically down-regulat ed early after cellular activation by CD40L and Staphylococcus aureus and m arkedly up-regulated as the cells apoptose. These findings identify a novel proapoptotic caspase adaptor protein.