(data not shown). These results suggest that Redox effects were not responsible for inhibition of apoptosis. Interest- ingly, Bax from pervanadate-treated cells also demon- strated a slight but reproducible decrease in mobility as seen by SDS-PAGE and immunoblotting. This was not due to direct tyrosine phosphorylation of Bax, as Bax was not detectable using anti-phosphotyrosine antibodies, nei- ther was Bax phosphorylation detected in immunoprecipi- tates from 32P-orthophosphate labeled cells. The cause for this mobility shift is, therefore, not yet clear. Pervanadate treatment had no effect on the distribution of Bcl-x or its mobility on SDS-PAGE. Together, these results indicate that protein tyrosine kinases are central in mediating ad- hesion-regulated subcellular localization of Bax and apop- tosis. Bax Localization and Conformation Is Regulated by pp125FAK Pp125FAK has previously been reported to regulate adhe- sion-dependent cell survival in serum starved fibroblasts (Hungerford et al., 1996; Ilic et al., 1998). To determine if inhibition of pp125FAK signaling in mammary epithelial cells resulted in apoptosis and altered exposure of the Bax BH3 epitope, its activity was inhibited by using dominant negative FAK constructs. A dominant negative pp125FAK, consisting of its carboxy-terminal region fused to glu- tathione-S-transferase (DN-FAK), contains the focal adhe- sion-targeting sequence of pp125FAK but lacks the kinase domain and the critical tyrosine phosphorylation site at Tyr- 397 required for interaction with src and PI 3-kinase. This construct has previously been shown to displace endoge- nous pp125FAK from the focal adhesions of fibroblasts and endothelial cells (Gilmore and Romer, 1996). To ascertain whether or not pp125FAK was required for survival in mammary epithelia, FSK-7 cells were trans- fected with either DN-FAK or pCDNA.3 alone, and the number of apoptotic cells determined 24 h later. DN-FAK induced almost five times the amount of apoptosis seen with vector alone, and this was inhibited with zVAD- fmk (Fig. 7 a). To examine whether DN-FAK regulated Bax conformation, transfected cells were immunostained using the BH3 proximal antibody (62M). DN-FAK– expressing cells that showed apoptotic morphology stained strongly with 62M, whereas there was little im- munoreactivity in nonexpressing cells with healthy nuclei (Fig. 7 b). 24 h after transfection, DN-FAK–expressing cells that had undergone apoptosis had mostly detached, and these cells were cytospun before immunostaining. Virtually all detached apoptotic cells stained positive both for DN-FAK and the 62M anti-Bax antibody. DN-FAK– expressing cells also stained strongly with the activated caspase 3 antibody (data not shown). Using transfections, we were unable to examine the time course of Bax epitope exposure after inhibition of pp125FAK signaling. Therefore, to address early events after inhibition of pp125FAK signaling, DN-FAK protein was microinjected into FSK-7 cells. Cells were then as- sayed at 1 and 5 h postinjection (Fig. 7 c). FSK-7 cells in- jected with DN-FAK showed a marked inhibition of phos- photyrosine staining in focal adhesions 1 h after injection compared with noninjected cells and to cells injected with the GST control (data not shown). At 5 h postinjection, there was an increase in the proportion of DN-FAK– injected cells with apoptotic morphology, whereas very few GST-injected cells showed abnormal morphology (Fig. 7 c). Not all the DN-FAK–injected cells became apoptotic after 5 h, possibly due to each cell receiving different doses of the fusion protein, as was found previously in endothe- lial cells and fibroblasts where the DN-FAK inhibitory ef- fect was highly dose dependent (Gilmore and Romer, 1996). We also examined exposure of the Bax BH3 epitope after DN-FAK injection. Immunostaining using the 62M antibody increased in a proportion of DN-FAK– injected cells within 1 h of injection (Fig. 7 d). This staining showed a similar pattern of staining to that seen in FSK-7 cells on poly-HEMA, with a punctate distribution. These results demonstrate that injection of DN-FAK protein in adherent mammary cells leads to rapid exposure of its BH3 epitope within 1 h, followed by the induction of apop- tosis in a high proportion of cells within 5 h. Thus in ad- herent cells, pp125FAK is required to maintain survival and inhibiting its function results in rapid Bax BH3 do- main exposure, followed by apoptosis. To determine whether Bax was required for DN-FAK– induced apoptosis, the BH3 proximal antibody 62M was coin- jected into FSK-7 cells with the fusion proteins. Cells were then examined 5 h post injection. Whereas DN-FAK–induced cell death when coinjected with nonspecific rabbit IgG, there was no loss of cells when injected together with 62M (Fig. 7 e). Thus, antibody binding near the critical BH3 domain required for apoptosis prevented Bax apoptotic function. Injection with either antibody (IgG or 62M) together with GST had no significant effect on apoptosis. These results provide evidence that the exposure of the Bax BH3 epitope is required for apoptosis induced by inhibiting pp125FAK signaling. Pp125FAK Mediates Apoptosis through PI 3-Kinase and pp60src Pp125FAK can activate a number of downstream signal- ing pathways (Hanks and Polte, 1997). PI 3-kinase and pp60src both bind to pp125FAK (Chen and Guan, 1994; Cobb et al., 1994) and have been implicated in survival sig- naling (Khwaja et al., 1997; McGill et al., 1997). To de- termine if pp125FAK mediated mammary cell survival through either PI 3-kinase or pp60src, DN-FAK was cotransfected with either the constitutively active p110 subunit of PI 3-kinase (p110CAAX) or with activated pp60src. Both active PI 3-kinase and pp60src blocked DN- FAK–induced apoptosis (Fig. 8 a). Previously, pp125FAK has been shown to promote survival of primary fibroblasts through a PKC- and p53-dependent pathway and not through a PI 3-kinase route (Ilic et al., 1998). To confirm that PI 3-kinase was required for MEC survival, FSK7 cells were transfected with a dominant negative p85 sub- unit (p85DSH2). This induced apoptosis as efficiently as DN-FAK (Fig. 8 a). We examined the exposure of the Bax BH3 domain in p85DSH2-transfected cells. Cells express- ing p85DSH2 stained strongly with the 62M antibody and showed apoptotic nuclei, whereas healthy untransfected cells were negative for Bax BH3 epitope exposure (Fig. 8 b). The p85DSH2-expressing cells that had detached after apoptosis were also examined after cytospinning, and Gilmore et al. Integrin Regulates Apoptosis through Bax Activation 439