A nucleotide binding site in caspase-9 regulates apoptosome activation.
ATP or dATP is a required activator of Apaf-1 for formation of the Apoptosome and thereby activation of caspase-9 (Csp9) [Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413]. Here we demonstrate that dATP or ATP may have an additional role in controlling Apaf-1-mediated Csp9 activation. In the presence of cytochrome c (CytC), dATP or ATP binds to Apaf-1 and triggers heptamerization of Apaf-1 leading to the activation of Csp9. At concentrations greater than 1 mM, dATP or ATP also functions as a negative regulator of apoptosis by binding to and inhibiting Csp9. The affinity labeling reagent, 3'-O-(5-fluoro-2,4-dinitrophenyl)-ATP (FDNP-ATP), was used to probe the binding of nucleotides to Csp9. Similar to ATP, but with a much more profound effect, FDNP-ATP binds to the full-length proCsp9 potently, with an IC(50) of approximately 5-11 nM. Neither ATP nor FDNP-ATP exhibits any effect on the prodomain-truncated enzyme DeltaproCsp9 or p18/p10. FDNP-ATP covalently labels proCsp9 with a stoichiometry of 1:1, resulting in DNP-ATP-proCsp9 that is incapable of forming a productive Apoptosome with Apaf-1. Activity assays show that ATP and dATP, but not ADP or AMP, bind to the processed Csp9 p35/p10. This nucleotide binding site might play an important and previously unrecognized role in regulating proCsp9 activation.
Chereau
David
D
Idun Pharmaceuticals, Inc., 9380 Judicial Drive, San Diego, California 92121, USA.
Zou
Hua
H
Spada
Alfred P
AP
Wu
Joe C
JC
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000345:Affinity Labels; D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D001665:Binding Sites; D053453:Caspase 9; D020169:Caspases / Q000737:chemistry* / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
C506592:APAF1 protein, human; D000345:Affinity Labels; D053477:Apoptotic Protease-Activating Factor 1; D003838:Deoxyadenine Nucleotides; D011506:Proteins; D011994:Recombinant Proteins; C057111:3'-O-(5-fluoro-2,4-dinitrophenyl)ATP ether; D000255:Adenosine Triphosphate; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases; C026600:2'-deoxyadenosine triphosphate