Role of Bax and pH in Death by Cytokine Withdrawal

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DESCRIPTION (provided by applicant): Gene products that regulate apoptosis are frequently mutated in cancer. Normally, apoptotic cell death is an essential part of lymphocyte development and maintenance, with the loss of a survival or trophic signal, such as a cytokine, as principal trigger for death. Hence, elucidation of novel regulatory proteins and metabolic changes during cytokine withdrawal is of vital importance for the understanding of the cell death process and more so for discovering new components mutated during carcinogenesis. To this end, the study of apoptosis induced by cytokine withdrawal in cytokine-dependent cell lines has proven successful. In initial studies, loss of cytokine signaling led to a rapid and transient alkalinization of the cytosol, mediated by the pH regulating protein, the sodium/hydrogen exchanger1 (NHE1) and p38 MAP kinase (MAPK). This rise in pH is proposed to cause the mitochondrial translocation of the pro-apoptotic protein, Bax, resulting in cell death. As the first aim, a two-part study is proposed to rigorously test the hypothesis that alkaline pH causes the movement of Bax to mitochondria by neutralizing charged amino acids in the termini. First, pH-independent modifications or indirect pH effects on Bax will be evaluated. Second, a series of Bax mutants will be made to target proposed pH sensitive sites and tested for mitochondrial translocation under apoptotic and non-apoptotic conditions. The expected outcome is to prove that Bax is a pH-responsive protein. In the second aim, the mediator of the pH rise, NHE1, will be evaluated for function during apoptosis. Previous work identified four candidate sites on NHE1 as targets for p38 MAPK phosphorylation. The importance of these sites will be determined by, first, performing p38 MAPK kinase assays using mutated GST-NHE1 fusion proteins as substrates for phosphorylation, and second, expressing full-length NHE1 mutant proteins in an NHE1-deficient cell line under apoptotic and non-apoptotic conditions. It is expected that mutant NHE1 expressing cells will not alkalinize, Bax will not translocate and death will be inhibited. Future aims include analysis of the alkalinization mechanism through NHE and elaboration of the role of Bax in mitochondrial damage. This work will be the foundation of an academic research program, as a tenure-tracked faculty, for the identification and application of novel apoptotic factors mutated in cancer from the unique perspective of pH-mediated apoptotic changes.