Bcl-2 Family Member Bfl-1/A1 Sequesters Truncated Bid to Inhibit Its Collaboration with Pro-apoptotic Bak or Bax

Abstract

Following caspase-8 mediated cleavage, a carboxylterminal fragment of the BH3 domain-only Bcl-2 family member Bid transmits the apoptotic signal from death receptors to mitochondria. In a screen for possible regulators of Bid, we defined Bfl-1/A1 as a potent Bid interacting protein. Bfl-1 is an anti-apoptotic Bcl-2 family member, whose preferential expression in hematopoietic cells and endothelium is controlled by inflammatory stimuli. Its mechanism of action is unknown. We find that Bfl-1 associates with both full-length Bid and truncated (t)Bid, via the Bid BH3 domain. Cellular expression of Bfl-1 confers protection against CD95-and Trail receptor-induced cytochrome c release. In vitro assays, using purified mitochondria and recombinant proteins, demonstrate that Bfl-1 binds full-length Bid, but does not interfere with its processing by caspase-8, or with its mitochondrial association. Confocal microscopy supports that Bfl-1, which at least in part constitutively localizes to mitochondria, does not impede tBid translocation. However, Bfl-1 remains tightly and selectively bound to tBid and blocks collaboration between tBid and Bax or Bak in the plane of the mitochondrial membrane, thereby preventing mitochondrial apoptotic activation. Lack of demonstrable interaction between Bfl-1 and Bak or Bax in the mitochondrial membrane suggests that Bfl-1 generally prevents the formation of a pro-apoptotic complex by sequestering BH3 domainonly proteins.

Multiple and synergistic deregulations of apoptosis-controlling genes in pancreatic carcinoma cells

“…Since it was shown (Hinz et al, 2000;Trauzold et al, 2001) that pancreatic tumour cell lines behave like type II cells in both, CD95-and TRAIL-mediated apoptosis, we did not only analyse the expression levels of caspases and DISC components, but also of proteins which participate in the mitochondrial apoptosis pathway (Werner et al, 2002). Therefore, we additionally analysed the expression of Bfl-1.…”

“…The observed strong down regulation of Bid in sensitive Colo357 cells is accompanied with nearly absence of the expression of Bcl-x L as well as Bfl-1. Bfl-1 is a protein of the Bcl-2 family, which has recently been shown to bind and inactivate truncated Bid (Werner et al, 2002). Thus no or very low expression of Bfl-1 in Colo357 cells in conjunction with very little amounts of Bcl-x L may account for the observed sensitivity of these cells to CD95-and TRAIL-mediated apoptosis.…”

Differential effects of Vav‐promoter‐driven overexpression of BCLX and BFL1 on lymphocyte survival and B cell lymphomagenesis

“…Twenty to thirty micrograms of total protein was loaded on 12% Bis‐Tris acrylamide gels and blotted on Amersham™ Hybond™—ECL nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). The following antibodies were used for protein detection: rabbit anti‐BFL1 (kindly provided by Jannie Borst 50), rat anti‐mouse A1 (WEHI, 6D6, 2 μg·mL −1 ) 51, rabbit anti‐BIM/BOD (Enzo, Farmingdale, NY, USA, polyclonal, ADI‐AAP‐330‐E, 0.2 μg·mL −1 ), rabbit anti‐MCL1 (polyclonal, Rockland, Pottstown, PA, USA, Cat# 600‐401‐394, 2.2 μg·mL −1 ), rabbit anti‐BCLX (54H6, Cell Signaling, Danvers, MA, USA, Cat# CS2764, 1 : 1000), mouse anti‐BCL2 (7/Bcl‐2, BD Biosciences, 0.5 μg·mL −1 ), mouse anti‐HA (HA.11, Covance, Princeton, NJ, USA, 1 : 1000), rabbit anti‐MYC (Y69, Abcam, Cambridge, UK, ab32072), rabbit anti‐VAV1 (Cell Signaling #2502, 1 : 1000), rabbit anti‐gamma‐H2A.X (Ser139, Cell Signaling #2577, 1 : 1000), rabbit anti‐GAPDH (14C10, Cell Signaling #2118, 1 : 5000), rabbit anti‐beta‐Actin (polyclonal, Cell Signaling #4967, 1:1000) and mouse anti‐HSP90 (F‐8, Santa Cruz, Dallas, TX, USA, Cat# sc‐13119, 0.2 μg·mL −1 ). All primary antibodies were diluted in 5% BSA in PBST and blots were incubated overnight at 4 °C.…”

Mechanism of action of Drosophila Reaper in mammalian cells: Reaper globally inhibits protein synthesis and induces apoptosis independent of mitochondrial permeability

“…Plasmid pMT2SM-Myc allows the protein to be amino-terminally Myc-tagged. 46 The ubiquitin ligase defective XIAP mutant (H467A) was made by site-directed mutagenesis of the human XIAP cDNA (histidine 467 changed to alanine) as previously described. 9 pCR3 vector encoding amino-terminally FLAGtagged human Survivin was provided by Dr. S Lens (The Netherlands Cancer Institute, Amsterdam, Netherlands).…”

“…Retroviral vector LZRS-Bcl-2-IRES-Zeo/pBR encoding human Bcl-2 and the corresponding empty vector have been described previously. 46 Plasmid pMT2SM encoding Myc-tagged human type I a PIP kinase was provided by Dr. N Divecha (The Netherlands Cancer Institute, Amsterdam, The Netherlands). All sequences were verified by dideoxynucleotide sequencing.…”