Protein kinase A regulates caspase-9 activation by Apaf-1 downstream of cytochrome c.
The cyclic AMP signal transduction pathway modulates apoptosis in diverse cell types, although the mechanism is poorly understood. A critical component of the intrinsic apoptotic pathway is caspase-9, which is activated by Apaf-1 in the apoptosome, a large complex assembled in response to release of cytochrome c from mitochondria. Caspase-9 cleaves and activates effector caspases, predominantly caspase-3, resulting in the demise of the cell. Here we identified a distinct mechanism by which cyclic AMP regulates this apoptotic pathway through activation of protein kinase A. We show that protein kinase A inhibits activation of caspase-9 and caspase-3 downstream of cytochrome c in Xenopus egg extracts and in a human cell-free system. Protein kinase A directly phosphorylates human caspase-9 at serines 99, 183, and 195. However, mutational analysis demonstrated that phosphorylation at these sites is not required for the inhibitory effect of protein kinase A on caspase-9 activation. Importantly, protein kinase A inhibits cytochrome c-dependent recruitment of procaspase-9 to Apaf-1 but not activation of caspase-9 by a constitutively activated form of Apaf-1. These data indicate that extracellular signals that elevate cyclic AMP and activate protein kinase A may suppress apoptosis by inhibiting apoptosome formation downstream of cytochrome c release from mitochondria.
Martin
Morag C
MC
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Scotland, United Kingdom.
Allan
Lindsey A
LA
Lickrish
Michelle
M
Sampson
Catherine
C
Morrice
Nick
N
Clarke
Paul R
PR
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002474:Cell-Free System; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000502:physiology*; D045304:Cytochromes c / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014981:Xenopus
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D011506:Proteins; D012694:Serine; D045304:Cytochromes c; D000242:Cyclic AMP; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases