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.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-04012007-204633 |
| Date | 14 April 2007 |
| Creators | Moser, Leta Ruth |
| Contributors | Elizabeth Yang, Sandra Zinkel |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-04012007-204633/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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