PRO-CASPASE-3 IS A MAJOR PHYSIOLOGICAL TARGET OF CASPASE-8

The apoptotic signal triggered by ligation of members of the death rec eptor family is promoted by sequential activation of caspase zymogens, We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rat es (k(cat)/K-m) of 8.7 x 10(5) and 2.8 x 10(5) M-1 s(-1), respectively , These rates are of sufficient magnitude to indicate direct processin g in vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, a nd specificities. The pattern and rate of caspase-3 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pa thway to activation, procaspase-9, was without consequence. Taken toge ther these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transm ission of the apoptotic signal generated by ligation of death receptor s.