Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B.
Cytototoxic T lymphocyte-induced apoptosis can occur either through the directed exocytosis of granzyme B and perforin or via ligation of Fas. Both pathways involve the activation of a family of cysteine proteinases, the caspases, that cleave substrates at aspartic acid and are themselves activated by cleavage at internal aspartate residues. Fas recruits caspase 8, which initiates the death program through the subsequent activation of caspase 3. Granzyme B can process both caspase 8 and 3 in vitro, suggesting that both Fas and granzyme B access the apoptotic program in the same way. Here we demonstrate that although the two mechanisms are similar, the events that lead to activation of caspase 3 can be distinguished in vivo on the basis of their sensitivities to both pharmacological and virus-encoded caspase inhibitors. In cytotoxic T lymphocytes-mediated death the initial cleavage event on caspase 3 is insensitive to benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (zVAD-fmk) inhibition in both mouse and human systems. During Fas-mediated death, however, activation of caspase 3 is completely inhibited to zVAD-fmk. In addition, the viral serpin SPI-2, a homologue of cytokine response modifier A (crmA), is an effective inhibitor of the Fas but not the granzyme pathway. Our results demonstrate that whereas Fas-mediated activation of caspase 3 requires an upstream caspase activity that is zVAD-fmk-sensitive, the initial cleavage of caspase 3 during granule-mediated cell death is insensitive to zVAD-fmk, suggesting that caspase 3 is cleaved directly by granzyme B in vivo.
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
E A
EA
Atkinson
M
M
Barry
A J
AJ
Darmon
I
I
Shostak
P C
PC
Turner
R W
RW
Moyer
R C
RC
Bleackley
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D053804:Granzymes; D006801:Humans; D019169:Jurkat Cells; D007939:Leukemia L1210 / Q000473:pathology; D051379:Mice; D009842:Oligopeptides / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C509213:GZMB protein, human; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases