the balance between apoptosis and survival, much like a rheostat (Yin et al., 1994). However, other data indicates that their pro- and anti-apoptotic functions are at least partially independent of their ability to interact with each other (Cheng et al., 1996; Simonian et al., 1996; Knudson and Korsmeyer, 1997; Simonian et al., 1997; St. Clair et al., 1997; Metcalfe et al., 1999). The crystal structure of Bcl-x suggests a potential to form membrane pores, and indeed Bcl-x, Bcl-2, and Bax all form pores in artificial bilayers (Muchmore et al., 1996; Minn et al., 1997). A number of Bcl-2 family members have a predicted transmembrane sequence at their carboxyl terminus, which localizes them to internal cell membranes including mitochondria and the ER. These proteins may act at the mitochondrial mem- brane by directly or indirectly inducing the release of proapoptotic molecules such as caspase 9 and cytochrome c (Kluck et al., 1997; Zou et al., 1997; Narita et al., 1998; Krajewski et al., 1999; Shimizu et al., 1999). Thus, although the mechanism of commitment to apoptosis is not yet fully understood, it may involve changes in mitochondrial per- meability that are regulated by Bcl-2 family proteins. Bax has been reported to be predominantly cytosolic in healthy cells, despite having a membrane-targeting carboxyl terminus. Bax translocates to mitochondria in a number of cell types after receipt of a death signal, such as cytotoxic insult (Hsu et al., 1997; Wolter et al., 1997; Goping et al., 1998; Gross et al., 1998; McGinnis et al., 1999). Deletion of the carboxy-terminal tail prevents Bax translocation and apoptosis after a death stimulus, suggesting that the regu- lation of its association with mitochondrial membranes may be a key stage in transduction of an apoptotic signal (Wolter et al., 1997). Indeed, enforced dimerization of Bax results in its constitutive targeting to mitochondria, result- ing in apoptosis in FL5.15 cells (Gross et al., 1998). Bcl-2 family proteins have a number of conserved sequences termed BH domains (for Bcl-2 homology domains). The BH1, BH2, BH3, and BH4 domains have critical functions in determining the pro- and anti-apoptotic properties of the various family members, and are involved in mediating interactions between them. The BH3 domain of Bax is a region of the molecule that is critically important for the apoptosis-inducing ability of the protein (Zha et al., 1996a; Cosulich et al., 1997; Wang et al., 1998). Mitochondria-tar- geted Bax requires a functional BH3 domain in order to induce apoptosis. At least two domains of Bax are there- fore required for its pro-apoptotic activity, a region that specifies its translocation to organelle membranes includ- ing mitochondria, and its BH3 domain. Cells adhere to the ECM through a family of hetero- dimeric transmembrane receptors, the integrins. Signaling pathways regulated by adhesion include tyrosine and serine/ threonine kinase activation, as well as changes in cellular pH, intracellular calcium and phospholipids (Schwartz et al., 1995). The tyrosine kinase pp125FAK (focal adhesion kinase) has been implicated as a central molecule for inte- grin-mediated signaling. Upon integrin ligation and clus- tering, pp125FAK becomes phosphorylated and activated, leading to its association with other kinases and adapter molecules, including PI 3-kinase, pp60src, p130CAS, paxil- lin and Grb2, which in turn leads to activation of down- stream pathways (Aplin et al., 1998; Schwartz and Baron, 1999). Pp125FAK is therefore poised to regulate adhesion- dependent cell phenotypes including survival (Gilmore and Romer, 1996; Zhao et al., 1998). Indeed, inhibition of pp125FAK by injection of anti-FAK antibodies or by ex- pression of a dominant negative FAK results in apoptosis in serum-deprived fibroblasts and cancer cell lines (Hunger- ford et al., 1996; Ilic et al., 1998), whereas expression of con- stitutively activated pp125FAK leads to the anchorage- independent survival of MDCK cells (Frisch et al., 1996). However, although pp125FAK is a candidate for regulat- ing adhesion-mediated survival, a link between pp125FAK and apoptosis regulatory proteins has not been identified. In this paper, we sought to define the mechanism by which adhesion to ECM regulates the apoptotic machin- ery. Since Bcl-2 family proteins have a central role in apop- tosis induced by a wide variety of different cellular insults, we investigated the possibility that their function is con- trolled by adhesion-mediated signaling. We found that the apoptotic protein Bax was largely present in the cytosol of adherent mammary epithelial cells. Detachment from ECM induced a rapid, but reversible, translocation of Bax to mitochondria concurrent with a conformational change resulting in the exposure of its BH3 domain. Bax translo- cation and BH3 epitope exposure occurred before com- mitment to apoptosis. We identified a critical role for ki- nase cascades in adhesion-regulated survival signaling and suggest that conformation of the Bax BH3 epitope is de- pendent on both pp125FAK and PI 3-kinase. Materials and Methods Cell Culture and Apoptosis Assays Primary mouse mammary cells, isolated from pregnant ICR mice (Pullan and Streuli, 1996), and FSK-7 cells (Kittrell et al., 1992), were grown in DMEM/F12 supplemented with 2% fetal calf serum, 5 ng/ml epidermal growth factor and 880 nM insulin. To assay detachment-induced apopto- sis, confluent cells were trypsinized and replated in whole medium onto dishes coated with polyhydroxyethylmethacrylate (poly-HEMA). In the case of inhibitor experiments, cells were preincubated with inhibitors 1 h before trypsinization and throughout incubation on poly-HEMA. Inhibitors were obtained from Calbiochem and used at the following concentrations: cycloheximide was used at 25 mg/ml, zVAD-fmk, and Ac-DEVD-cmk at 100 mM, herbimycin A at 1 mM, and wortmannin at 1 mM; pervanadate was used at 1 mM from sodium orthovanadate and hydrogen peroxide (Sigma), and treated with catalase (Sigma) to quench unreacted hydrogen peroxide. After incubation for various times, cells were cytospun onto poly- sine slides (Merck), and fixed in 2% paraformaldehyde. To quantify apop- tosis, nuclear morphology was examined after staining cells with 4 mg/ml Hoescht 33258 (Molecular Probes). To analyze DNA integrity after incuba- tion on poly-HEMA, cells were washed in PBS and lysed in 100 mM NaCl, 10 mM EDTA, 25 mM Tris-Cl, pH 8.0, 0.65% SDS, and 500 mg/ml protein- ase K. Lysates were extracted with phenol/chloroform (1:1). DNA was ethanol precipitated, treated with RNase and separated on a 1.5% agarose gel containing ethidium bromide. Protein Extraction and Immunoblotting For fractionation studies, cells were washed once in PBS before resus- pending in hypotonic buffer (10 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-Cl, pH 7.6, 1 mM NaFl, and 100 mM sodium orthovanadate, containing pro- tease inhibitors) and lysed in a Dounce homogenizer before addition of 53 isotonic buffer (525 mM mannitol, 172 nM sucrose, 12.5 mM Tris-Cl, pH 7.6, and 2 mM EDTA). Cytosolic and membrane fractions were then centrifuged at 100,000 g. Equivalent amounts of protein were separated by SDS-PAGE and immunoblotted (Metcalfe et al., 1999). For cross-linking experiments, after incubations, cells were washed in PBS twice before in- cubating with 2.5 mM disuccinimidyl suberate (Pierce) in 200 mM manni- tol, 70 mM sucrose, 1 mM EDTA, and 10 mM Hepes, pH 7.5, for 30 min. The reaction was stopped by adding Tris-Cl, pH 7.6, to 20 mM. The total The Journal of Cell Biology, Volume 149, 2000 432