Phosphorylation-dependent Akt cleavage in neural cell in vitro reconstitution of apoptosis

Fleur François, Mark L. Grimes

Research output: Contribution to journalArticlepeer-review

Abstract

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 reactivates this key mediator of survival signaling, ensuring that the execution pathway will prevail.

Original languageEnglish
Pages (from-to)1773-1776
Number of pages4
JournalJournal of Neurochemistry
Volume73
Issue number4
DOIs
StatePublished - 1999

Keywords

  • Akt/protein kinase B
  • Apoptosis
  • Caspase
  • In vitro reconstitution
  • Phosphorylation

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