Ca2+-induced inhibition of apoptosis in human SH-SY5Y neuroblastoma cells:degradation of apoptotic protease activating factor-1 (APAF-1)

During apoptotic and excitotoxic neuron death, challenged mitochondria rele ase the pro-apoptotic factor cytochrome c. In the cytosol, cytochrome c is capable of binding to the apoptotic protease-activating factor-1 (APAF-1). This complex activates procaspase-9 in the presence of dATP, resulting in c aspase-mediated execution of apoptotic neuron death. Many forms of Ca2+-med iated neuron death, however, do not lead to prominent activation of the cas pase cascade despite significant release of cytochrome c from mitochondria. We demonstrate that elevation of cytosolic Ca2+ induced prominent degradat ion of APAF-1 in human SH-SY5Y neuroblastoma cells and in a neuronal cell-f ree apoptosis system. Loss of APAF-1 correlated with a reduced ability of c ytochrome c to activate caspase-3-like proteases. Ca2+ induced the activati on of calpains, monitored by the cleavage of full-length alpha -spectrin in to a calpain-specific 150-kDa breakdown product. However, pharmacological i nhibition of calpain activity indicated that APAF-1 degradation also occurr ed via calpain-independent pathways. Our data suggest that Ca2+ inhibits ca spase activation during Ca2+-mediated neuron death by triggering the degrad ation of the cytochrome c-binding protein APAF-1.