Sequential activation of three distinct ICE‐like activities in Fas‐ligated Jurkat cells

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Abstract
ICE family proteases have been implicated as important effectors of the apoptotic pathway, perhaps acting hierarchically in a protease cascade. Using cleavage of endogenous protease substrates as probes, three distinct tiers of ICE‐like activity were observed after Fas ligation in Jurkat cells. The earliest cleavage detected (30 min) was of fodrin, and produced a 150 kDa fragment. The second phase of cleavage (50 min) involved PARP, U1‐70kDa and DNA‐PKcs, all substrates of the CPP32‐like proteases. Lamin B cleavage was observed during the third cleavage phase (90 min). Distinct inhibition profiles obtained using a panel of peptide‐based inhibitors of ICE‐like proteases clearly distinguished the three different cleavage phases. These studies provide evidence for a sequence of ICE‐like proteolytic activity during apoptosis. The early fodrin cleavage, producing a 150 kDa fragment, identifies an ICE‐like activity proximal to CPP32 in Fas‐induced Jurkat cell apoptosis.