Genetics of Apoptosis

exposed BH3 domain, while the BH3 domain is buried in antiapoptotic proteins.
Exposure of the BH3 domain is believed to enable its hydrophobic face to interact
with the groove formed by BH1, 2, and 3 domains in a heterodimerization partner.
As the solved structures of Bid and Bax contain buried BH3 domains, they are
classified as latent proapoptotic proteins. Activating steps, caspase-mediated cleavage
in the former case and a conformational change dependent on tBid in the latter case,
are predicted to expose the BH3 domain. Proteolytic removal of the NH 2 -terminus
of Bcl-2 and Bcl-xL, by caspase-3, generates proapoptotic versions of these proteins
(Cheng et al., 1997a; Clem et al., 1998; Kirsch et al., 1999; Basañez et al., 2001).
The NH 2 -terminal α-helix forms an undercarriage for the BH3 helix; thus, removing
this portion of the protein may untether the BH3 domains. However, there are also
examples of function switching not explained by proteolytic unraveling of a compact
protein structure. Wang, N.S. et al. (2001) reported that transient transfection of
Bcl-2 in human embryonic kidney 293 and breast cancer MDA-MB-468 cells triggers
apoptosis despite mutation of Asp^34 at the caspase cleavage site, preventing caspase
cleavage. Even stranger was the report by Middleton et al. (1996) of a Bax survival
function in growth factor-dependent neuronal cells. It is hard to avoid the conclusion
that antiapoptotic and proapoptotic BH proteins have much in common, with
intracellular targeting, susceptibility to proteolysis, and perhaps post-translational
modifications, such as phosphorylation, providing their quite different cellular
phenotypes, at least most of the time.

3.

Location, location, location

The early debates over subcellular localization of Bcl-2 have quieted now with the
recognition of Bcl-2 effects that can be attributed to specific sites within the cell.
Bcl-2 is an integral membrane protein associated with outer mitochondrial membrane
and endoplasmic reticulum (Chen-Levy and Cleary, 1990; Monaghan et al., 1992).
There is little evidence for a soluble form of Bcl-2. While there remain some questions
concerning Bcl-2 distribution at inner mitochondrial or cristal membranes (Lombardi
et al., 1997; Motoyama et al., 1998; Gotow et al., 2000), much of the Bcl-2
fractionating with mitoplast preparations probably represents adherent contact sites
between the inner and outer membranes (de Jong et al., 1994). Nguyen et al. (1993)
showed that the COOH-terminal hydrophobic domain of Bcl-2 functions as a
mitochondrial targeting sequence, with much lower efficacy for ER protein import.
Other antiapoptotic proteins have homologous COOH-terminal domains and
appear to have similar intracellular distributions (Gonzalez-Garcia et al., 1994; Akgul
et al., 2000; Duriez et al., 2000; Lee et al., 2001; O’Reilly et al., 2001).

MAKING SENSE OF THE BCL-2 FAMILY OF APOPTOSIS REGULATORS 63