Calcium binding of ARC mediates regulation of caspase 8 and cell death

Apoptosis repressor with CARD (ARC) possesses the ability not only to block activation of Caspase 8 but to modulate caspase-independent mitochondrial events associated with cell death. However, it is not known how ARC modulates both caspase-dependent and caspase-independent cell death. Here, we report that ARC is a CA(2+)-dependent regulator of Caspase 8 and cell death. We found that in CA(2+) overlay and Stains-all assays, ARC protein bound to CA(2+) through the C-terminal proline/glutamate-rich (P/E-rich) domain. ARC expression reduced not only cytosolic CA(2+) transients but also cytotoxic effects of thapsigargin, A23187, and ionomycin, for which the CA(2+)-binding domain of ARC was indispensable. Conversely, direct interference of endogenous ARC synthesis by targeting ARC enhanced such CA(2+)-mediated cell death. In addition, binding and immunoprecipitation analyses revealed that the protein-protein interaction between ARC and Caspase 8 was decreased by the increase of CA(2+) concentration in vitro and by the treatment of HEK293 cells with thapsigargin in vivo. Caspase 8 activation was also required for the thapsigargin-induced cell death and suppressed by the ectopic expression of ARC. These results suggest that calcium binding mediates regulation of Caspase 8 and cell death by ARC.