Integrin-mediated Survival Signals Regulate the Apoptotic Function of Bax through Its Conformation and Subcellular Localization Andrew P. Gilmore,* Anthony D. Metcalfe,* Lewis H. Romer,‡ and Charles H. Streuli* *School of Biological Sciences, University of Manchester, Manchester, M13 9PT, UK; ‡Departments of Cell Biology and Anatomy, Pediatrics, and Anesthesiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Abstract. Most normal cells require adhesion to extra- cellular matrix for survival, but the molecular mecha- nisms that link cell surface adhesion events to the intra- cellular apoptotic machinery are not understood. Bcl-2 family proteins regulate apoptosis induced by a variety of cellular insults through acting on internal mem- branes. A pro-apoptotic Bcl-2 family protein, Bax, is largely present in the cytosol of many cells, but redis- tributes to mitochondria after treatment with apopto- sis-inducing drugs. Using mammary epithelial cells as a model for adhesion-regulated survival, we show that detachment from extracellular matrix induced a rapid translocation of Bax to mitochondria concurrent with a conformational change resulting in the exposure of its BH3 domain. Bax translocation and BH3 epitope expo- sure were reversible and occurred before caspase acti- vation and apoptosis. Pp125FAK regulated the confor- mation of the Bax BH3 epitope, and PI 3-kinase and pp60src prevented apoptosis induced by defective pp125FAK signaling. Our results provide a mechanistic connection between integrin-mediated adhesion and apoptosis, through the kinase-regulated subcellular dis- tribution of Bax. Key words: apoptosis • Bax • mammary • adhesion • pp125FAK Introduction Most normal cells require adhesion to extracellular matrix (ECM)1 for cell growth, differentiation and survival. Ad- hesion dependence permits cell differentiation and growth only when the cell is in its correct environment within the organism. Cells that become displaced from their appropri- ate locations are deleted by apoptosis, a highly regulated process by which unwanted or damaged cells are destroyed without inducing an inflammatory response (Meredith et al., 1993; Frisch and Francis, 1994). Apoptosis is tightly controlled and its deregulation leads to degenerative con- ditions or to the survival of abnormal cells, contributing to the development of tumors. In the mammary gland, lumi- nal epithelial cells are dependent upon adhesion to ECM for survival (Boudreau et al., 1995; Pullan et al., 1996; Far- relly et al., 1999; Streuli and Gilmore, 1999). Primary mam- Address correspondence to Charles Streuli, School of Biological Sciences, University of Manchester, 3.239 Stopford Building, Oxford Road, Manchester M13 9PT, UK. Tel.: 144 161 275 5576. Fax: 144 161 275 7700. E-mail: cstreuli@man.ac.uk 1Abbreviations used in this paper: BH, Bcl-2 homology; BrdU, bro- modeoxyuridine; DN-FAK, dominant-negative FAK; DSS, disuccinimidyl suberate; ECM, extracellular matrix; PI 3-kinase, phosphatidylinositol 3-kinase; pp125FAK, focal adhesion kinase; polyHEMA, polyhydroxy- ethylmethacrylate. mary epithelial cells show a specific requirement for a laminin-rich basement membrane for survival, and un- dergo apoptosis over several days if they are cultured on collagen I (Pullan et al., 1996). Laminin acts as a survival ligand through b1 integrins and cooperates with signals from insulin to suppress mammary epithelial apoptosis (Farrelly et al., 1999). However, the molecular mechanisms that link adhesion events at the cell surface to the intracel- lular apoptotic machinery are not understood. Apoptosis induced by a wide variety of factors, from growth factor starvation to cytotoxic drugs, occurs as a de- fined sequence of events. The apoptotic stimulus or insult must initially be sensed and transduced to the apoptotic machinery, but only after commitment to apoptosis does the execution phase occur through caspase activation. Members of the Bcl-2 family appear to function at a piv- otal point in the decision process where cells become irre- versibly committed to die. Some members of this family, including Bcl-x, Bcl-2, Bcl-w, and Mcl-1 promote cell sur- vival, whereas others, such as Bax, Bak, Bad, Bik, Bid, Bok, and Bim promote apoptosis (Adams and Cory, 1998). These molecules form both homo- and heterodimers, and one possible mechanism for their control on apoptosis is that the relative proportions of their interactions regulate ª The Rockefeller University Press, 0021-9525/2000/04/431/15 $5.00 The Journal of Cell Biology, Volume 149, Number 2, April 17, 2000 431–445 http://www.jcb.org 431