The Bcl-2 Family

Introduction

In C. elegans, ced-3 and ced-4 were identified as genes essential for programmed cell death, while ced-9 was found to be a regulator of cell death by preventing apoptosis. The first mammalian homolog for ced-3 was described in 1988 as Bcl-2 which is involved in B-cell lymphomas (Vaux and Adams, 1988, Nature 335: 440). Bcl-2 transfected B cells were shown to be resistant towards apoptosis normally induced in B cells by IL-3 withdrawal: for the first time it was shown that the pathway toward tumorigenesis depends not only on the ability to escape growth control but also depends on the ability to prevent apoptosis.

The members of the family

So far, 15 mammalian family members were identified, which were divided into three subfamilies:

1. Bcl-2 subfamily (pro-survival): Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1;
2. Bax subfamily (pro-apoptotic): Bax, Bak and Bok;
3. BH3 subfamily (pro-apoptotic): Bad, Bid, Bik, Blk, Hrk, BNIP3 and BimL;

Additionally, several Bcl-2 homologs have been identified in viruses, among others the adenovirus oncoprotein E1B-19K.

Some basic features

The bcl-2 gene codes for a 25 kDa protein. The C terminal 21 amino acids encode a stretch of hydrophobic amino acids that are important in membrane docking: Bcl-2 resides on the cytoplasmic face of the mitochondrial outer membrane, the nuclear envelop, and the endoplasmic reticulum. Deletion of the C terminus does not abrogate Bcl-2 survival function! Most Bcl-2 homologs have this hydrophobic C terminal domain, though they not necessarily are located on membranes but are cytosolic (e.g. Bax).
When homologs of Bcl-2 have been identified, it became apparent that the Bcl-2 family can be defined by the presence of conserved motifs known as Bcl-2 homology domains (BH1 to BH4). While Bcl-2 and its most similar prosurvival homologs Bcl-XL and Bcl-w contain all four BH domains, the other pro-survival members contain at least BH1 and BH2. While the members of the Bax subfamily contain BH1, BH2 and BH3, and resemble Bcl-2 fairly closely, the seven mammalian members of the BH3 subfamily possess only the central short (9 - 16 residue) BH3 domain and are unrelated to any known protein. The BH3 subfamily members may well represent the physiological antagonists of the pro-survival proteins, since programmed cell death in C. elegans requires EGL-1 (the one non-mammalian BH3 family member) and Bid was reported to link caspase-8 activity to cytochrome c release.

Pro- and anti-apoptotic family members can heterodimerize: the BH1, BH2 and BH3 domains of an anti-apoptotic member (e.g. Bcl-XL) form a hydrophobic cleft to which a BH3 amphipathic alpha-helix can bind (Sattler et al., 1997, Science, 275: 983). This BH3 cleft coupling, reminiscent of ligand-receptor engagement, may account for all dimerization within the family. Heterodimerization is not required for pro-survival function (contrary to early indications) but is essential for the pro-apoptotic activity in the BH3 subfamily. The Bax subfamily members do not depend essentially on heterodimerization, but possibly have an independent cytotoxic impact.

Mechanisms of Action

Bcl-2 and Bcl-XL bind to Apaf-1
A central checkpoint of apoptosis is the activation of Caspase-9 by mitochondria. The BH4 domain of Bcl-2 and Bcl-XL can bind to the C terminal part of Apaf-1 (to the CED-4 like part and the WD-40 domain), thus inhibiting the association of Caspase-9 with Apaf-1 (Hu et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 4386-4391; Huang et al., 1998, EMBO, 17(4): 1029-1039; Pan et al., 1998, J. Biol. Chem., 273(10): 5841-5845). This process seems to be conserved from nematodes to humans since in C. elegans CED-9 binds to CED-4, preventing it from binding and activating CED-3.

Bcl-2 family members regulate Cytochrome c release
The pro-survival proteins also seem to maintain organelle integrity since Bcl-2 directly or indirectly prevents the release of cytochrome c from mitochondria (Yang et al, 1997, Science, 275: 1129-1132). On the other hand, the pro-apoptotic BH3 subfamily member BID was reported to mediate the release of cytochrome c (without evoking mitochondrial swelling and permeability transition). Interestingly, BID is able to bind to pro-apoptotic members of the Bcl-2 family (e.g. Bax) as well as to pro-survival members Bcl-2 and Bcl-XL (Luo et al., 1998, Cell, 94: 481-490).

Bax maybe involved in Caspase-Independent Death
The BH1 and BH2 domains of Bcl-2 family members (Bcl-2, Bcl-XL and Bax) resemble membrane insertion domains of bacterial toxins: hypothetically they can form pores in organelles such as mitochondria, what at least was demonstrated in lipid bilayers in vitro. In yeast - which lack Bcl-2 like proteins, CED-4 and caspases - Bax and Bak were shown to induce cell death, while Bcl-2 can protect, apparently by preventing mitochondrial disruption (Green and Reed, 1998, Science, 281: 1309). Bax and Bax-like proteins might mediate caspase-independent death via channel-forming activity, which would promote the mitochondrial