BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax

Abstract

Bcl-2 was isolated from the t(14;18) chromosomal breakpoint in follicular B-cell lymphoma. Bcl-2 has the unique oncogenic role of extending cell survival by inhibiting a variety of apoptotic deaths. An emerging family of Bcl-2-related proteins share two highly conserved regions referred to here as Bcl-2 homology 1 and 2 (BH1 and BH2) domains (Fig. 1). This includes Bax which heterodimerizes with Bcl-2 and when overexpressed counteracts Bcl-2. We report here that site-specific mutagenesis of Bcl-2 establishes the two domains as novel dimerization motifs. Substitution of Gly 145 in BH1 domain or Trp 188 in BH2 domain completely abrogated Bcl-2's death-repressor activity in interleukin-3 deprivation, gamma-irradiation and glucocorticoid-induced apoptosis. Mutations that affected Bcl-2's function also disrupted its heterodimerization with Bax, yet still permitted Bcl-2 homodimerization. These results establish a functional role for the BH1 and BH2 domains and suggest Bcl-2 exerts its action through heterodimerization with Bax.

Bcl-xS and Bax induce different apoptotic pathways in PC12 cells

“…The results showed that the mI-3 and the DC22 mutants are e ective in this regard. It was previously shown that the mI-3 mutation causes Bcl-2 to lose its capacity for heterodimerization with Bax (St. Clair et al, 1997;Yin et al, 1994) and Bid (Wang et al, 1996). The ability of the mI-3 Bcl-2 mutant to inhibit Bcl-x Sinduced cell death thus demonstrates that this mutation does not eliminate the anti-apoptotic e ect of Bcl-2, at least with respect to apoptosis induced by Bcl-x S .…”

“…Mutations within the BH1 domain of Bcl-2 [G145A(mI-3) and G145E (mI-4)] have been shown to eliminate the Bcl-2 death repressor activity in several apoptotic systems, and to disrupt Bcl-2 heterodimerization with Bax (Yin et al, 1994). The Bcl-2DC22 mutant, on the other hand, reportedly provides partial protection from cell death and is capable of interacting with Bax (Oltvai et al, 1993).…”

“…pCMV ± SEAP was obtained from Dr Pradier (Rhone-Poulenc Rorer). The vectors pcDNA3Bcl-2, pcDNA3Bcl-2 mI-3 (G145A), pcDNA3Bcl-2 mI-4 (G145E) and pcDNA3Bcl-2DC22 (with deletion of 22 amino acids from the C-terminal, which contains the signal anchor sequence) were generated by inserting cDNAs of human wild-type Bcl-2 (Seto et al, 1998) and its mutant cDNAs (Oltvai et al, 1993;Yin et al, 1994) into the EcoRI site of pcDNA3. The Bax expression vector pcDNA3HA muBax contains mouse Bax cDNA (Oltvai et al, 1993) and was generated in Dr Korsmeyer's laboratory (Washington University, St. Louis, MO, USA).…”

t(14;18), a journey to eternity

“…Mutant nonfunctional BCL2 still dimerizes with wild-type BCL2, suggesting that BCL2 and BCL-X L must dimerize with BAX to display their anti-apoptotic activity. 85,88 However, the amino acid residues within BH1 and BH2 that are involved in maintaining the antiapoptotic activity of BCL2 and BCL-X L are partially different. Moreover, the cell death protective function of BCL-X L has been demonstrated in the absence of heterodimerization with BAX or BAK.…”

Complete activation of Bax by a single site mutation

“…Other pro-apoptotic members, such as Bid and Bad, possess only the BH3 domain (Adams and Cory, 1998). Mutational analyses indicated that the BH1 and BH2 domains of Bcl-2 and Bcl-X L are required for heterodimerization with Bax to antagonize Bax's pro-apoptotic activity (Yin et al, 1994). The BH3 domain of Bax was shown to be essential for dimerization (both homo-and hetero-) and for its apoptosis-inducing ability (Zha et al, 1996).…”