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Phosphorylation‐Dependent Akt Cleavage in Neural Cell In Vitro Reconstitution of Apoptosis
Author(s) -
François Fleur,
Grimes Mark L.
Publication year - 1999
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1999.731773.x
Subject(s) - phosphorylation , cleavage (geology) , apoptosis , in vitro , protein kinase b , microbiology and biotechnology , neural cell , chemistry , cell , biology , biochemistry , paleontology , fracture (geology)
: 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.