Eukaryotic cells respond to extracellular stimuli by transmitting intracellular instructions via signalling pathways to coordinate appropriate responses. Mitogen-activated protein kinase (MAPK) pathways are often used to transmit these instructions to regulate gene expression, where Ternary Complex Factors (TCFs) are among their nuclear targets. Elk-1 is the founding member of the TCF family of transcription activators. The mechanism of function and regulation of Elk-1 has been extensively studied, therefore providing a paradigm for signal-induced transcription. The activity of Elk-1 is influenced by Post-Translational Modifications (PTMs), such as phosphorylation and sumoylation. Elk-1 ubiquitylation has also been reported in vitro, however little work has been done on this modification of Elk-1. This thesis sought to reveal the mechanism of regulation and function of Elk-1 ubiquitylation. Elk-1 was demonstrated to be both monoubiquitylated and polyubiquitylated in vitro and in cells. Using size exclusion chromatography and the dominant negative nedd8 conjugating E2 enzyme Ubc12, several features of the Elk-1 specific E3 ligases have been revealed in vitro. In cells, ternary complex formation was shown to be important for monoubiquitylation. Furthermore monoubiquitylated Elk-1 is diminished following ERK-mediated phosphorylation, hence activation, in response to mitogen stimulation. It was also demonstrated that an Elk-1 derivative that exhibits strong monoubiquitylation level also exhibits a reduced capability to transactivate gene expression at the Serum Responsive Element (SRE), indicating a negative role of monoubiquitylation on Elk-1 transcriptional ability.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:548927 |
| Date | January 2011 |
| Creators | Chow, Kam Yuen |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/12035/ |
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