cases, apoptosis occurs even in the presence of serum growth factors, including insulin and EGF, demonstrating the primal nature of the ECM-derived survival signal. Pp125FAK represents an excellent candidate signaling protein for transducing adhesion-mediated survival sig- nals. It has been shown to be a key apoptosis regulator in several cell types including fibroblasts, human cancer cell lines, and MDCK epithelial cells (Frisch et al., 1996; Hun- gerford et al., 1996; Ilic et al., 1998; Xu et al., 1998; Sonoda et al., 1999). Our work now implicates an essential role for pp125FAK in mammary cell survival since both microin- jected DN-FAK protein and transfected DN-FAK expres- sion vectors induce apoptosis rapidly. Although we cannot totally discount the possibility that DN-FAK disrupts cell adhesion, and thus induces apoptosis, we think this is un- likely. First, DN-FAK did not disrupt focal adhesions when microinjected into fibroblasts and primary endothe- lial cells, although it did block downstream signaling (Gilmore and Romer, 1996). Second, the number of de- tached cells resulting from DN-FAK transfection was re- duced by treatment with zVAD-fmk, indicating that detach- ment was due to apoptosis rather than disruption of adhesion by DN-FAK. Third, activated mutants of PI 3-kinase and src also reduced the number of cells detaching after DN-FAK transfection. Together these observations suggest that de- tachment follows apoptosis in DN-FAK–expressing cells and not vice versa. A variety of downstream effectors may propagate sur- vival signals from pp125FAK, as it lies in a nodal position for effecting integrin-dependent cellular responses and can potentially act through PI 3-kinase, pp60src, as well as adapter molecules such as Grb2, paxillin and p130CAS (Chen and Guan, 1994; Schlaepfer et al., 1994; Schaller and Parsons, 1995; Cary et al., 1996; Chen et al., 1996; Cary et al., 1998; Thomas et al., 1998). One candidate signaling kinase is PI 3-kinase, which regulates apoptosis in kidney epithelial cells as well as in neuronal and hematopoietic cells (Minshall et al., 1996; Dudek et al., 1997; Khwaja et al., 1997; Philpott et al., 1997; Vaillant et al., 1999). A po- tential role for PI 3-kinase in adhesion-mediated survival signaling comes from experiments where the expression of constitutively activated PI 3-kinase as well as one of its substrates, protein kinase B, protects MDCK cells from apoptosis in suspension (Khwaja et al., 1997), and where adhesion to fibronectin directly results in the accumula- tion of PI 3-kinase lipid products as well as p85-dependent protein kinase B activity (King et al., 1997). In the mam- mary system we have previously demonstrated that apoptosis is induced by pharmacological inhibitors of PI 3-kinase (Farrelly et al., 1999), and now show that cell death results from expression of the dominant negative p85 sub- unit of PI 3-kinase, whereas apoptosis induced by DN- FAK is suppressed by the constitutively active p110 sub- unit of PI 3-kinase. Since adhesion to ECM leads to the association of PI 3-kinase with pp125FAK and its activation in several cell types (Chen and Guan, 1994; Chen et al., 1996; King et al., 1997), including mammary epithelial cells (our unpublished data), it would seem to be a likely down- stream effector of pp125FAK-mediated survival signaling. However, the plethora of binding sites for signaling mol- ecules on FAK suggests that it may regulate survival through different mechanisms in different cell types. This is borne out at both the cellular and molecular levels, as detachment of fibroblasts from ECM, for example, does not lead to rapid apoptosis induction as is the case for epi- thelia (Frisch and Francis, 1994). In serum-starved rat syno- vial fibroblasts, DN-FAK induced apoptosis, but this was not reversed by activating the PI 3-kinase pathway (Ilic et al., 1998). In this cell type, pp125FAK acted through a p53-dependent pathway involving the l/i isoform of pro- tein kinase C and phospholipase A2. Further experiments showing that PI 3-kinase inhibition did not induce apop- tosis suggests that fibroblasts may be independent of PI 3-kinase for survival. Thus, whereas PI 3-kinase is important in epithelia, it may not be a primary determinant of adhe- sion-mediated survival in other cell types such as synovial fibroblasts. Other pp125FAK-mediated pathways than PI 3-kinase may also be involved with epithelial cell survival. Constitu- tively active pp125FAK can suppress apoptosis in MDCK cells detached from ECM, but only if its kinase domain and its major autophosphorylation site, Y-397, are intact (Frisch et al., 1996). Y-397 binds src family kinases in addi- tion to PI 3-kinase. This association has been proposed to regulate the phosphorylation of cellular substrates through the recruitment of src to focal adhesions (Schaller et al., 1999). In mammary cells we found that activated pp60src also suppressed the apoptotic effects of DN-FAK. Furthermore, transformation by src has been implicated in reducing the sensitivity of fibroblasts to detachment- induced apoptosis (McGill et al., 1997). Whether or not PI 3-kinase and pp60src trigger similar or independent down- stream pathways leading to apoptosis suppression is not yet clear. Pp125FAK effects on cell proliferation, through Erk activation, appear to be regulated by Y-397 binding to src and fyn kinase, whereas its regulation of migration also requires an association with p130CAS through one of two carboxy-terminal proline rich domains (Cary et al., 1998; Zhao et al., 1998). Recently, it has been suggested that pp125FAK can activate Jun amino-terminal kinase through a mechanism not requiring pp125FAK kinase ac- tivity, but involving an interaction with paxillin through its extreme carboxy-terminal region (Igishi et al., 1999). Thus, the many interactions of pp125FAK with down- stream signaling pathways might permit several mecha- nisms for regulating apoptosis. Linking Integrin Signals to the Apoptosis Machinery Adhesion provides an essential anti-apoptotic signal for epithelial cells, yet the mechanism by which it feeds into the apoptotic machinery has not been elucidated. We demonstrate in this paper that whatever the proximal sur- vival pathway regulated by pp125FAK, adhesion regulates apoptosis through a Bax-dependent mechanism. Previously, integrin-mediated adhesion has been shown to upregulate expression of Bcl-2 in CHO cells, which was proposed to lead to survival according to the rheostat model (Zhang et al., 1995). However, in mammary epithelia Bcl-2 is absent and we have seen no changes in the levels of ex- pression of Bax or Bcl-x either after detachment from ma- trix or during culture on different ECM substrata (Pullan et al., 1996). Thus, at least in mammary epithelia, altered ra- tios of these proteins do not seem to regulate apoptosis. The Journal of Cell Biology, Volume 149, 2000 442