Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis

Kai Huang; Jingjing Zhang; Katelyn L. O'Neill; Channabasavaiah B. Gurumurthy; Rolen M. Quadros; Yaping Tu; Xu Luo

doi:10.1074/jbc.M115.711051

Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis*

From the ^‡Eppley Institute for Research in Cancer and Allied Diseases,
^¶Department of Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, and
^‖Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, Nebraska 68198-7696,
^**Department of Pharmacology, Creighton University School of Medicine, Omaha, Nebraska 68178, and
^§Xiangya Hospital, Central South University, Changsha 410008, China

↵2 To whom correspondence should be addressed: Eppley Inst. for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-7696. Tel.: (402)-559-4643; Fax: (402)-559-3739; E-mail: xuluo{at}unmc.edu.

↵1 Both authors contributed equally to this work.

Abstract

The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo. In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant Bid^D60E and a BH3 defective mutant Bid^G94E failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis.

Footnotes

↵* This work was supported in part by the Nebraska Center for Cellular Signaling, which is funded by an Institutional Development Award (IDeA) from NIGMS, National Institutes of Health under Grant P30 GM106397, and by National Institutes of Health Grant 1R21CA193271 (to Y. T.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.