Sequential activation of three distinct ICE-like activities in Fas-ligated Jurkat cells.
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.
Greidinger
E L
EL
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Miller
D K
DK
Yamin
T T
TT
Casciola-Rosen
L
L
Rosen
A
A
D000911:Antibodies, Monoclonal / Q000276:immunology; D034961:Antigens, Nuclear; D017209:Apoptosis; D002352:Carrier Proteins / Q000378:metabolism; D020170:Caspase 1; D053148:Caspase 3; D020169:Caspases; D018797:Cell Cycle Proteins; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting; D034921:Lamin Type B; D034882:Lamins; D008840:Microfilament Proteins / Q000378:metabolism; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000276:immunology / Q000378:metabolism*; D013049:Spectrin
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D002352:Carrier Proteins; D020170:Caspase 1; D053148:Caspase 3; D020169:Caspases; D018797:Cell Cycle Proteins; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D034921:Lamin Type B; D034882:Lamins; D008840:Microfilament Proteins; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D011065:Poly(ADP-ribose) Polymerases; D017346:Protein Serine-Threonine Kinases; D017412:Ribonucleoprotein, U1 Small Nuclear; D019014:fas Receptor; D013049:Spectrin; C050045:NUMA1 protein, human; C490688:SNRNP70 protein, human; C497263:PRKDC protein, human; C505407:CASP3 protein, human