The tumor suppressor gene, Bax, plays a critical role in tumor progression through regulating cell apoptosis. Mutations on the BAX gene often... Show moreThe tumor suppressor gene, Bax, plays a critical role in tumor progression through regulating cell apoptosis. Mutations on the BAX gene often result in silencing its expression and the loss of pro-death ability. However, there is a unique Bax isoform, BaxΔ2, recently discovered in these Bax mutated cancer cells. BaxΔ2 isoform shows higher pro-apoptotic activity than Baxα. Unlike the parental Baxα, BaxΔ2 does not target mitochondria and forms aggregates in cytosol. There is a unique 10-amino-acid peptide in the N-terminus of BaxΔ2 protein possible function as a special signal. Two serines in this region are predicted as potential phosphorylation sites for regulation of the protein activity. To test this hypothesis, we mutated both serines (SS) into non-phosphorylatable alanines (AA) by site-directed mutagenesis approach. Both BaxΔ2 wild type (BaxΔ2-SS) and mutants (BaxΔ2-AA) were tagged with GFP, which allows us to monitor the protein expression and cellular localization in live cells. Here, we found that the distribution patterns of BaxΔ2-AA and BaxΔ2-SS were similar and appeared as aggregates in cytosol. BaxΔ2-AA mutant also possessed the similar pro-apoptotic activity with BaxΔ2-SS wild type. These results suggested that the two serines in BaxΔ2 unique oligopeptide might not play a critical role in BaxΔ2 localization and pro-death activity under the current ectopic expression conditions. Further study is needed to have better understanding of phosphorylation in contribution to unique behavior of BaxΔ2. M.S. in Biology, July 2014 Show less
