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J Biol Chem, Vol. 273, Issue 33, 21261-21266, August 14, 1998

Cytotoxic T Lymphocyte-assisted Suicide
CASPASE 3 ACTIVATION IS PRIMARILY THE RESULT OF THE DIRECT ACTION OF GRANZYME B

Eric A. Atkinson, Michele Barry, Alison J. Darmon, Irene Shostak, Peter C. Turner, Richard W. Moyer, and R. Chris Bleackley

From the  Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and  Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610-0266

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.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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