Characterization of a novel proapoptotic caspase-2- and caspase-9-binding protein.
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.
Bonfoco
E
E
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA. ebonfoco@scripps.edu
Li
E
E
Kolbinger
F
F
Cooper
N R
NR
D048868:Adaptor Proteins, Signal Transducing; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001402:B-Lymphocytes / Q000166:cytology / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D023201:CD40 Ligand / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053143:Caspase 2; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013211:Staphylococcus aureus / Q000502:physiology; D014158:Transcription, Genetic; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; C434482:MZB1 protein, human; D011994:Recombinant Proteins; D023201:CD40 Ligand; D000255:Adenosine Triphosphate; C506433:CASP9 protein, human; C506435:Casp9 protein, rat; D053143:Caspase 2; D053453:Caspase 9; D020169:Caspases