Cleavage of Bid by Executioner Caspases Mediates Feed Forward Amplification of Mitochondrial Outer Membrane Permeabilization during Genotoxic Stress-induced Apoptosis in Jurkat Cells

Shary N. Shelton,John D. Robertson,Mary E. Shawgo

doi:10.1074/jbc.m809392200

Abstract

The extent to which the BH3-only protein Bid is important for intrinsic (mitochondria-mediated) apoptotic cell death induced by genotoxic stress remains controversial. In the present study, we examine this issue using a panel of gene-manipulated Bax-deficient Jurkat T-lymphocytes. Cells stably depleted of Bid were far less sensitive than control-transfected cells to etoposide-induced apoptosis. In particular, drug-induced Bak activation, cytochrome c release, loss of mitochondrial membrane potential, and caspase activation were all decreased in cells lacking Bid. Reconstitution experiments using recombinant proteins and permeabilized Bid-deficient cells demonstrated that truncated Bid (tBid), but not full-length Bid, potently induced Bak activation and the release of cytochrome c. Further, caspase-8-deficient Jurkat cells efficiently cleaved Bid and were sensitive to drug-induced apoptosis. By comparison, Apaf-1-deficient cells, as well as cells overexpressing full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP, failed to cleave Bid in response to genotoxic stress. These data suggest that tBid plays an important regulatory role in the execution of DNA damage-induced cytochrome c release and apoptosis. However, the fact that cleavage of Bid to tBid is mediated by executioner caspases suggests that a self-amplifying feed forward loop involving caspases, Bid, and mitochondria may help determine irreversible commitment to apoptosis.

Highlights

Execution of apoptosis is mediated by a family of cysteinedependent aspartate-specific proteases
Evidence in the literature indicates that cells lacking either Bak or Bax exhibit only subtle defects in mitochondrial outer membrane permeabilization (MOMP), whereas doubly deficient cells are often found to be highly resistant to mitochondria-mediated apoptosis [7, 8]
Because we had observed that etoposide-induced apoptosis was decreased in Bid-deficient Jurkat cells, we evaluated mitochondrial events that characteristically define MOMP, such as the dimerization and activation of Bak or Bax, the release of intermembrane space proteins, and the loss of ⌬⌿

Summary

Introduction

Execution of apoptosis is mediated by a family of cysteinedependent aspartate-specific proteases (caspases). Western blot analysis indicated that both Bid-deficient clones were less sensitive than of Jurkat whole-cell lysates obtained at 6 h following incubation control-transfected cells to etoposide-induced apoptosis with with etoposide (10 ␮M) revealed that Bid had been cleaved to the degree of resistance paralleling the extent to which Bid tBid. Drug-induced cleavage of Bid to tBid was accompanied by expression had been suppressed (Fig. 2B).

Results

Conclusion