| dc.description.abstract | Although the signaling mechanisms of the apoptotic pathway have been extensively studied, there is still much left unknown. A key regulator protein, 14-3-3, is known to bind and protect BAD phosphorylation sites serine 112 and 136, sequestering BAD in the cytoplasm. Under conditions of cell death, 14-3-3 becomes loose and exposes the phosphorylated S112 site to phosphatases, promoting the sequential dephosphorylation of BAD at sites 112, 136, and 155, thereby inducing apoptosis. The examination of the BAD-14-3-3 complex under conditions of cell death is of acute interest to this thesis. Three possibilities were examined that may aid in the dissociation of 14-3-3 to BAD under conditions of cell death: 1) interaction of an unknown binding partner 2) novel BAD modification or phosphorylation 3) 14-3-3 modification or phosphorylation. Methods used in these investigations included: Mass Spectrometry, kinase assays, pulldown experiments, and western blots. Several novel binding partners to BAD were identified in either a condition of cell life or cell death, including the following: p38, CDC2, Beclin-1, BAX, cytochrome c1 oxidase, and voltage-dependent anion-selective channel proteins 1, 2, and 3. 32P kinase assays suggested that p38 phosphorylates a novel site on BAD, serine 6. The phosphorylation state of BAD S128 was observed 3-7 hours after cell death stimulation and was independent of p38 activation. Under cell life and death conditions, no new phosphorylation sites or monomeric changes on 14-3-3 were observed; p38 was concluded not to phosphorylate 14-3-3. | |
