Figure 7. Mammary epithelial cell apoptosis and Bax BH3 epitope exposure is regulated by pp125FAK. (a) FSK-7 cells were trans- fected with either DN-FAK or vector alone, in the presence of either 100 mM zVAD or carrier alone. 24 h post transfection, the number of apoptotic cells were determined by Hoechst staining. (b) DN-FAK–transfected FSK-7 cells were immunostained 24 h posttransfec- tion for GST and Bax using anti-Bax 62M. Both adherent cells and cells that had detached after apoptosis were stained. DN-FAK– expressing cells showing Bax staining were apoptotic, whereas all nonexpressing cells showed healthy nuclei and were negative for Bax immunoreactivity (examples of healthy nontransfected cells are indicated by arrows). All detached cells were Bax positive and had apop- totic nuclei. (c) Adherent FSK-7 cells were microinjected with either 3 mg/ml GST or GST-tagged DN-FAK protein. Cells were fixed 1 and 5 h postinjection, followed by immu- nostaining for GST to identify injected cells. Cells were stained with Hoechst 33258 and the percentage of apoptotic cells that were GST-positive were scored based on nuclear condensation/fragmentation. (d) Adherent FSK-7 cells were microinjected with GST or DN-FAK protein and fixed 1 h postinjection. Cells were double immunostained for GST and Bax using the anti-Bax 62M antibody. Note the appearance of Bax staining in the cell microinjected with DN-FAK, but absence of staining in cells injected with GST alone. (e) Adherent FSK-7 cells were microinjected with either GST or DN-FAK protein, together with either anti-Bax 62M or rabbit IgG. In- jected cells were identified by immunostaining with anti-GST, and the number of apoptotic cells determined. these all showed Bax epitope exposure (Fig. 8 b). Simi- larly, treatment of FSK-7 cells with the PI 3-kinase inhibi- tor wortmannin resulted in apoptosis and translocation of Bax to the insoluble fraction (data not shown). Bax epitope exposure was inhibited in cells that had been cotransfected with DN-FAK and either p110CAAX or src, consistent with the hypothesis that signaling through PI 3-kinase and src linked pp125FAK with suppression of Bax activation (Fig. 8 c). Together with the fibroblast study (Ilic et al., 1998), these results indicate that pp125FAK might suppress apoptosis using cell type–specific signaling path- ways. We suggest that in mammary epithelial cells, the acti- vation of pp125FAK by integrin-mediated adhesion to ECM may act through downstream kinases such as PI 3-kinase to regulate the conformation and subcellular distribution of Bax, thereby controlling apoptotic fate. Discussion ECM is an important determinant for epithelial cell sur- vival (Frisch and Francis, 1994; Boudreau et al., 1995; Khwaja et al., 1997; Farrelly et al., 1999), but until now the components that link adhesion to the apoptotic machinery have not been identified. In this paper, we demonstrate that detachment of epithelial cells from ECM results in re- versible changes in the conformation and localization of Bax before the onset of apoptosis. We have identified early decision making events in this process, as ECM inter- actions suppress apoptosis through signaling pathways ac- tivated by the integrin-associated kinase pp125FAK. Our results suggest that pp125FAK, through PI 3-kinase and pp60src, maintains Bax in a conformation that prevents its relocation to mitochondria. Bax translocation is upstream The Journal of Cell Biology, Volume 149, 2000 440