The role of bad phosphorylation status and binding partners in promoting apoptosis.

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.