Bfl-1/A1 functions, similar to Mcl-1, as a selective tBid and Bak antagonist

Abstract

The prosurvival Bcl-2-family member Bfl-1/A1 is a transcriptional target of nuclear factor-kappaB (NF-kappaB) that is overexpressed in many human tumors and is a means by which NF-kappaB inhibits apoptosis, but its mode of action is controversial. To better understand how Bfl-1 functions, we investigated its interaction with proapoptotic multidomain proteins Bax and Bak, and the BH3-only proteins Bid and tBid. We demonstrate that in living cells Bfl-1 selectively interacts with Bak and tBid, but not with Bax or Bid. Bfl-1/Bak interaction is functional as Bfl-1 suppressed staurosporine (STS)-induced apoptosis in wild-type and Bax-deficient cells, but not in Bak-/- cells. We also show that Bfl-1 blocks tumor necrosis factor-alpha (TNFalpha)-induced activation of Bax indirectly, via association with tBid. C-terminal deletion decreased Bfl-1's interaction with Bak and tBid and reduced its ability to suppress Bak- and tBid-mediated cell death. These data indicate that Bfl-1 utilizes different mechanisms to suppress apoptosis depending on the stimulus. Bfl-1 associates with tBid to prevent activation of proapoptotic Bax and Bak, and it also interacts directly with Bak to antagonize Bak-mediated cell death, similar to Mcl-1. Thus, part of the protective function of NF-kappaB is to induce Mcl-1-like activity by upregulating Bfl-1.

Figure 1

Bfl-1 and Bfl-1ΔC suppress TNα-induced cyto C release. Immunofluorescence of MCF-7 cells transfected with GFP (a–f), GFP-Bfl-1 (g–n) or –Bfl1ΔC (o–v) with (d–f, k–n; s–v) or without (a–c, g–j, o–r) TKFα treatment for 8 h, using an anti-cyto C antibody (c, f, i, m, q, u). Cells were also analysed for GFP fluorescence (b, e, h, l, p, t). Cyto C, cytochrome C; GFP, green fluorescent protein; TNFα, tumor necrosis factor-α.

Figure 2

Bfl-l and BfI-1ΔC suppress TNFα-induced Bax conformational activation. Immunofluorescence of activated Bax in MCF-7 cells transfected with GFP (a–d), GFP-Bfl-1 (e–h) or -Bfl-1ΔC (i–l) with (c and d, g and h, k and l) or without (a and b, e and f, i and j) TNFα treatment, using an anti-BaxNT antibody (b, d, f, h, j, l). Cells were also analysed for GFP fluorescence (a, c, e, g, i, k). Asterisks mark GFP-Bfl-1 or -Bfl-1 AC-positive cells that failed to show Bax activation.

Figure 3

Bfl-1 and Bfl-1ΔC can interact with overexpressed Bax, but fail to associate with endogenous Bax regardless of Bax activation status. (a) Co-immunoprecipitation of GFP-Bfl-1 or -Bfl-1ΔC with overexpressed FLAG-Bax in CHAPS extracts from cotransfected HeLa cells. (b) Immunoprecipitation of 2×Myc-Bfl-1 or -Bfl-1ΔC with endogenous Bax in HeLa cells treated with TNFα plus CHX or CHX alone, using anti-Bax-loop antibody and immunoblotting with anti-Myc or -BaxN20 antibody. Lys: 1/10 input lysate. CHX, cycloheximide; GFP, green fluorescent protein; TNFα, tumor necrosis factor-α.

Figure 4

Bfl-1 selectively interacts with endogenous Bak but not Bax and suppresses apoptosis in wild type and Bax-deficient iBMK cells, but not in Bak-deficient iBMKs. (a) Co-immunoprecipitation of endogenous Bax or Bak with GFP-Bfl-1, endogenous Bak or 2×Myc-Bfl-1 in HeLa cells with or without TNFα/CHX treatment to activate Bax and Bak. NT: non-transfected. A line demarcates different exposures of the same blot. (b) Co-immunoprecipitation of endogenous Bax or Bak with 2×Myc-Bfl-1 compared with FLAG-Mcl-1 was performed as in panel (a). (c) Immunoprecipitation of endogenous Bak with 2×Myc-Bfl-1, -Bfl-1ΔC or FLAG-Mcl-1. (d) Immortalized wild-type, Bak−/−, Bax−/− and Bax−/−Bak−/− double-knockout iBMKs transfected with EGFP (hatched), 2×Myc-Bfl-1 (black), 2×Myc-Bfl-1ΔC (white) or FLAG-Mcl-1 (dotted) were treated with STS for 24 h or left untreated. Average survival in three experiments was determined by Trypan blue exclusion and normalized to transfection efficiency. CHX, cycloheximide; GFP, green fluorescent protein; iBMK, immortal baby mouse kidney; STS, staurosporine; TNFα, tumor necrosis factor-α.

Figure 5

Bfl-1 and Bfl-1ΔC selectively interact with endogenous tBid but not Bid in TNFα-treated cells and suppress tBid-induced apoptosis. (a) Immunoprecipitation/western blot of GFP-Bfl-1 or -Bfl-1ΔC with overexpressed Bid-Myc or tBid-Myc in HeLa cells. (b) Co-immunoprecipitation of endogenous Bid or tBid with 2×Myc-Bfl-1 or -Bfl-1ΔC in HeLa cells treated with TNFα/CHX or CHX alone. A longer exposure is shown. Asterisks mark the positions of endogenous Bid and tBid; a nonspecific band appeared above Bid in the lysates. (c) GFP-Bfl-1 or -Bfl-1ΔC suppress tBid-induced cell death in MCF-7 cells cotransfected with tBid-FLAG or pcDNA3, together with CMV-β-gal. The average of three experiments is shown. (d) Model illustrating Bfl-1’s selective interaction with tBid and Bak to suppress TNFα- and Bak-dependent apoptosis, and how its mode of action resembles that of Mcl-1. CHX, cycloheximide; GFP, green fluorescent protein; TNFα, tumor necrosis factor-α.