The BCL-2 family pro-apoptotic proteins have tumor suppressive functions and are important for conferring chemosensitivity to anti-neoplastic drugs. In animal cells, various apoptotic stimuli activate expression of specific BH3-only proteins. These proteins induce apoptogenic conversion of BH123 proteins (BAX and BAK) that leads to cell death. The molecular mechanisms of how BH3-only proteins activate BH123 proteins are unknown. BIK, the founding member of the BH3-only family mediates its pro-apoptotic activity primarily through BAX in human epithelial cancer cells. We will investigate a major hypothesis that interaction of BIK with BCL-2 family anti-apoptotic members such as BCL-2 results in functional activation of BAX and specific sequences in BAX determine functional activation BAX. Aim 1 will identify the BAX sequences that are important for BAX activation and function from a library of random and targeted mutants of Bax and by studying the effect of various Bax isoforms. Functionally altered Bax mutants will be studied to determine the effects on subcellular localization, membrane insertion, oligomerization and structural properties. Aim 2 will investigate the specificity of various human BH3-only proteins for the human BH123 proteins BAX and BAK in normal human cells depleted for these proteins or in mouse double knockout cells reconstituted with the human proteins. Aim 3 will investigate if the pro-apoptotic activity of BIK is linked to its ability to interact with BCL-2 family anti-apoptosis proteins and test a potential model for BAX activation through BIK interaction with BH1234 proteins. Aim 4 will investigate the hypothesis that BIK-induced apoptosis is initiated at the endoplasmic reticulum and amplified in the mitochondria. Specifically, the potential role of BIK in disruption of intracellular calcium homeostasis and the involvement of the cysteine protease calpain in apoptosis signaling by BIK will be investigated. The proposed studies should illuminate the apoptotic pathway by which BIK activates the apoptotic machinery through BAX. The knowledge gained would be valuable in modulating the activity of BAX in cancer and in degenerative diseases.