Phosphorylation-dependent Akt cleavage in neural cell in vitro reconstitution of apoptosis.
Neuronal apoptotic execution uses a cytochrome c-dependent caspase activation mechanism that is conserved in other cell types. Phosphatidylinositol 3-kinase and its downstream effector, Akt/protein kinase B, appear to control this mechanism and govern the life/death decision. We have developed a cell-free system using cytosol from human neuroblastoma (SY5Y) cells that reconstitutes biochemical features of neuronal apoptosis. In the presence of cytochrome c and ATP, caspase-9 and -3 were activated, which initiated chromatin condensation and DNA cleavage in rat pheochromocytoma (PC12) nuclei. Akt was cleaved in reactions where caspase-3 was activated and its cleavage was prevented by the caspase inhibitor DEVD-aldehyde. The phosphatase inhibitors orthovanadate and okadaic acid prevented catalytic processing and activation of caspase-3 and digestion of Akt and partially inhibited cleavage of caspase-9. Caspase-dependent destruction of Akt irreversibly inactivates this key mediator of survival signaling, ensuring that the execution pathway will prevail.
François
F
F
Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.
Grimes
M L
ML
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D002843:Chromatin / Q000187:drug effects / Q000648:ultrastructure; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008959:Models, Neurological; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology / Q000502:physiology; D016716:PC12 Cells; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D014407:Tumor Cells, Cultured
D002843:Chromatin; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; C494918:AKT1 protein, human; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C505407:CASP3 protein, human; C506433:CASP9 protein, human; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases