Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosisby the mitochondria-dependent caspase activation

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitute s a pivotal signaling pathway in cytokine- and stress-induced apoptosis. Ho wever, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-vaI-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-indu ced cell death, suggesting that caspases are required for ASK1-induced apop tosis. Overexpression of ASK1 Delta N, a constitutively active mutant of AS K1, induced cytochrome c release from mitochondria and activation of caspas e-9 and caspase-3 but not of caspase 8-like proteases. Consistently, caspas e-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1 -induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9- dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release , which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 acti vation and cell death induced by ASK1. ASK1 thus appears to execute apoptos is mainly by the mitochondria-dependent caspase activation.