Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation.
The essential upstream steps in granzyme B-mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B-resistant Bcl-2-overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B-treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.
Sutton
V R
VR
Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Melbourne 8006, Australia.
Davis
J E
JE
Cancilla
M
M
Johnstone
R W
RW
Ruefli
A A
AA
Sedelies
K
K
Browne
K A
KA
Trapani
J A
JA
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D001854:Bone Marrow Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D053804:Granzymes; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D011499:Protein Processing, Post-Translational; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D015398:Signal Transduction; D019014:fas Receptor / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; C494815:Bid protein, mouse; D002352:Carrier Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; C509213:GZMB protein, human; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases; D020169:Caspases