The adapter protein apoptotic protease-activating factor-1 (Apaf-1) is proteolytically processed during apoptosis.
Apoptotic protease-activating factor-1 (Apaf-1), a key regulator of the mitochondrial apoptosis pathway, consists of three functional regions: (i) an N-terminal caspase recruitment domain (CARD) that can bind to procaspase-9, (ii) a CED-4-like region enabling self-oligomerization, and (iii) a regulatory C terminus with WD-40 repeats masking the CARD and CED-4 region. During apoptosis, cytochrome c and dATP can relieve the inhibitory action of the WD-40 repeats and thus enable the oligomerization of Apaf-1 and the subsequent recruitment and activation of procaspase-9. Here, we report that different apoptotic stimuli induced the caspase-mediated cleavage of Apaf-1 into an 84-kDa fragment. The same Apaf-1 fragment was obtained in vitro by incubation of cell lysates with either cytochrome c/dATP or caspase-3 but not with caspase-6 or caspase-8. Apaf-1 was cleaved at the N terminus, leading to the removal of its CARD H1 helix. An additional cleavage site was located within the WD-40 repeats and enabled the oligomerization of p84 into a approximately 440-kDa Apaf-1 multimer even in the absence of cytochrome c. Due to the partial loss of its CARD, the p84 multimer was devoid of caspase-9 or other caspase activity. Thus, our data indicate that Apaf-1 cleavage causes the release of caspases from the apoptosome in the course of apoptosis.
Lauber
K
K
Department of Internal Medicine I, University of Tübingen, D-72076 Tübingen, Germany.
Appel
H A
HA
Schlosser
S F
SF
Gregor
M
M
Schulze-Osthoff
K
K
Wesselborg
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D016685:Mitomycin / Q000494:pharmacology; D008954:Models, Biological; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D020449:Repetitive Sequences, Amino Acid; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D019014:fas Receptor / Q000378:metabolism
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D011506:Proteins; D019014:fas Receptor; D016685:Mitomycin; D005047:Etoposide; D000255:Adenosine Triphosphate; C505407:CASP3 protein, human; C505514:CASP6 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D019311:Staurosporine