Bax is a pro-apoptotic member of the Bcl-2 protein family that plays a pivotal role in the apoptotic response of cancer cells to chemotherapeutic drugs and radiation. A conformational change in the Bax protein appears to be required for the cytosolic Bax to move to the outer mitochondrial membrane (OMM) where it forms oligomers or clusters that cause mitochondrial dysfunction and apoptotic cell death. To seek Bax-interacting partners, we and others have independently cloned a novel Bax-binding protein termed Bif-1 and SH3GLB1, respectively, by yeast two hybrid screens using Bax as the bait. Ectopic expression of Bif-1 in FL5.12 cells promotes IL-3 deprivation-induced Bax conformational change, caspase activation, and apoptotic cell death. Conversely, loss of Bif-1 suppresses these events in response to cytotoxic stress and promotes tumorigenicity of HeLa cells. It is not clear how Bif-1 is involved in the process of Bax conformational change, but there are interesting indications suggesting that Bif-1 has membrane-deforming activity. Bif-1 binds to intracellular membranes and deforms liposomes into tubules. Moreover, Bax-mediated lipidic pore formation occurs through a mechanism sensitive to intrinsic membrane curvature. We therefore hypothesize that Bif-1 - mediated OMM deformations facilitate and/or stabilize Bax oligomerization and integration into OMM during apoptosis. In addition, our results that Bif-1 binding to Bax is induced by apoptotic stimuli suggest a possibility that the ability of Bif-1 to activate Bax is regulated by post-translational modifications such as phosphorylation. Indeed, our preliminary studies showed that Bif-1 is phosphorylated in cells and the levels of Bif-1 phosphorylation are decreased during apoptosis. Furthermore, we have generated Bif-1-deficient mice and found that homozygotes (bif-1-/-) displayed seizures, ataxia, and early mortality postnatally. To investigate Bif-1 function in Bax activation and to explore the physiological roles of Bif-1 in mammalian development and tissue homeostasis, we propose the following Specific Aims: 1) To determine the role of Bif-1 in Bax oligomerization and insertion into OMM, cytochrome c release, and apoptosis; 2) To determine the molecular basis and functional significance of Bif-1 phosphorylation; 3) To explore the in vivo functions of Bif-1 by characterizing Bif-1 knockout mice.
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