The long term goal of the proposed research is to develop apoptosis inducing therapy that can be targeted for chemoprevention of cancer caused by aberrant expression of the anti-apoptosis oncogene Bc12 ( B cell lymphoma/leukemia-2). Taxol and other microtubule-targeting drugs can trigger Bc12 phosphorylation at G2-M phase of the cell cycle. By site directed mutagenesis, the phosphorylation sites were identified to be Serine-70 & Serine-87 residues of Bc12 protein. Our preliminary studies employing this unique phosphorylation-deficient mutant clearly indicate that Bc12 phosphorylation is detrimental to its anti-apoptotic function. Interestingly, sequences surrounding both Serine-70 and 87 constitute consensus motifs for substrate of MAP/ERK2 kinase. Precisely, the clear understanding of the loss of Be12 function by phosphorylation as well as the identification of the kinase/phosphatase pathway regulating function of Bc12 are crucial at this stage of our investigation. The generation of the phosphorylation-deficient mutant will now be extremely helpful for the accomplishment of five Specific Aims proposed here.
In Specific Aim 1, the interdependence of Bc12 phosphorylation and G2-M arrest will be tested in G2-M population of normal dividing cells transfected with wild and phospho-deficient mutant Bc12. Due to phosphorylation, any change in subcellular localization of phospho-Bc12 will be assessed by employing phospho-Bc12 specific antibody.
Specific Aim 2 will test the hypothesis that phospho-Bc12 fails to form dimer with its proapoptotic partner Bax by co- immunoprecipitation and protein-protein interaction using GST-fusion column.
In Specific Aim 3, the experiments pursuing the characterization of the kinase and phosphatase regulating Bc12 phosphorylation will be undertaken. In order to search for in vivo phospho-deficient form of Bc12, a variety of malignant cells (not responsive to microtubule targeting drug induced Bc12 phosphorylation and apoptosis) will be screened by RT-PCR in Specific Aim 4. The generation of transgenic mice expressing phospho-deficient mutant Bc12 in Specific Aim 5 will be helpful for in depth functional analysis of Bc12 phosphorylation. In all, these studies will offer new insights into the molecular mechanism of Bc12 function as well as provide valuable information for a potential genetic screen of apoptosis related human diseases.
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