Instead, we find that cell-ECM interactions control Bax translocation between the cytosol and mitochondria. Al- though Bax is a pro-apoptotic death effector, we argue that adhesion-dependent signaling maintains Bax in the cytosol where it is unable to trigger apoptosis. In response to lack of integrin-mediated survival signals, Bax translo- cates to a cellular environment where it can initiate apop- tosis. Although Bax has been found constitutively associ- ated with internal cell membranes in some cell types (Desagher et al., 1999), GFP-Bax chimeras had a predomi- nantly cytosolic distribution in Cos-7 cells and in other cell types Bax was cytosolic until receipt of a death stimulus, at which point redistribution to mitochondria occurred (Wolter et al., 1997; Goping et al., 1998; Gross et al., 1998). In common with all but the BH3-only proteins in the Bcl-2 family, Bax has a hydrophobic carboxy-terminal tail of z20 amino acids. This tail contains a mitochondrial-tar- geting domain, and directs unrelated peptides to mito- chondria (Nguyen et al., 1993; Wolter et al., 1997; Ne- chushtan et al., 1999). Targeting of Bax to internal membranes appears to be critical for its ability to induce apoptosis, as deletion of the hydrophobic tail abrogates the ability of Bax to induce apoptosis not only in our ex- periments with mammary epithelia but also in Cos-7 cells and L929 fibroblasts (Wolter et al., 1997). Thus, regulation of Bax targeting to internal cell membranes may be a criti- cal regulatory step in apoptosis. Concomitant with the translocation of Bax from the cy- tosol to mitochondria is a conformational change that may be important for its regulation. In hematopoietic cells the amino terminus of Bax is inaccessible to a monoclonal an- tibody when the protein is in a cytosolic form but be- comes exposed during translocation associated with an apoptotic stimulus (Hsu et al., 1997). In our studies an an- tibody recognizing an epitope just amino-terminal to its BH3 domain becomes exposed after cell detachment from ECM or upon expression of DN-FAK in adherent cells. The Bax BH3 domain is necessary for the induction of apoptosis, as peptides resembling this domain have apop- tosis-inducing ability, and substitution of the Bcl-2 BH3 region with that of Bax converts Bcl-2 into a pro-apoptotic protein (Hunter and Parslow, 1996; Cosulich et al., 1997). This would indicate that masking of the BH3 domain is re- quired to maintain survival in healthy cells, but its expo- sure after an apoptotic stimulus may be an essential part of the commitment process. This suggestion is supported by our own experiments, where coinjection of DN-FAK with the Bax BH3 proximal antibody inhibited the apoptotic ef- fect of the fusion protein. The result directly implicates control of BH3 domain exposure with adhesion-mediated survival signals. The related molecule Bak, shows similar changes in antibody reactivity after treatment of lym- phoma cells with DNA damaging agents (Griffiths et al., 1999). However, in this case, although Bak undergoes a conformational change to expose an amino-terminal do- main during the early stages of apoptosis commitment, it is constitutively associated with mitochondria. Conforma- tional changes associated with the exposure of the death- inducing BH3 domain may therefore be a common mecha- nism of activation within the Bcl-2 family in response to a wide variety of cellular insults. It not yet resolved how Bax conformation and subcellu- lar localization are controlled, but both its amino and car- boxyl termini have been implicated. Deletion of the amino terminus results in dysregulated translocation of in vitro– translated Bax to purified mitochondria, whereas replac- ing the carboxyl terminus of Bax with the corresponding sequence of Bcl-2, which is constitutively targeted to mem- branes, also results in constitutive targeting of Bax (Go- ping et al., 1998). A further point of regulation may be through phosphorylation, although it is unclear whether Bax can be directly phosphorylated. We did not detect phosphorylated Bax in mammary cells radiolabeled with 32P-orthophosphate, even though other phosphoproteins were readily identified. The BH3-only protein Bad, for ex- ample, appears to be suppressed in its apoptosis-inducing ability by IL-3–mediated phosphorylation of S-112 and S-136, which provides binding sites for the chaperone pro- tein 14-3-3. After IL-3 withdrawal, Bad becomes dephos- phorylated and translocates to mitochondria (Zha et al., 1996b; Datta et al., 1997). Bax expressed in E. coli can be phosphorylated on serine residues by both protein kinase A and MAP kinase (Lewis et al., 1998), although it is not yet known whether this has relevance to Bax regulation in vivo. We found that tyrosine kinase inhibitors induced Bax translocation to mitochondria and BH3 domain epitope exposure in mammary epithelia. Likewise, the protein ki- nase C inhibitor staurosporine, induced relocalization of Bax in Cos-7 cells (Wolter et al., 1997). It will therefore be important to determine whether kinase-mediated Bax lo- calization occurs through direct or indirect means. It remains possible that Bax function is regulated by a chaperone. Intriguingly, in vitro–translated Bax was un- able to associate with mitochondria unless it had been ex- posed to a cell lysate derived from apoptotic cells (Goping et al., 1998). Bax does not contain consensus 14-3-3–bind- ing sites, and in our own experiments Bax did not associate with 14-3-3 (unpublished data), which would implicate the involvement of other proteins. One possibility is the BH3- only protein, Bid, although this is unlikely in mammary epi- thelia as we have been unable to detect it in FSK-7 cells (un- published data). Bid resides in the cytoplasm of healthy cells and does not induce apoptosis, but after cleavage by caspase 8, which is activated by death receptors such as TNFR and FAS, Bid was found to bind Bax and induce exposure of the Bax amino-terminal region (Green, 1998; Desagher et al., 1999). This suggests that in some cell types, Bid can regulate Bax activity. Recently, caspase 8 has been shown to be activated after MDCK cell detach- ment from ECM (Frisch, 1999; Rytomaa et al., 1999). However, it is not clear whether Bax activation is depen- dent upon caspase 8 since zVAD, which inhibits caspase 8, blocked neither Bax translocation to mitochondria in mammary cells in our studies, nor the appearance of Bax immunoreactivity in HeLa cells treated with staurosporine (Desagher et al., 1999). Many questions remain to be an- swered regarding the precise control of pro-apoptotic Bcl-2 proteins, and it is quite possible that this regulation shows distinct tissue specific variations. In summary, we have now identified one potential mech- anism whereby adhesion can regulate commitment to apop- tosis in epithelial cells. Integrin-mediated positional control is required for maintaining homeostasis, and defective inte- grin signals lead normal cells to commit apoptosis. By alter- Gilmore et al. Integrin Regulates Apoptosis through Bax Activation 443