The transcription factor ELK-1 is associated with numerous cellular processes, notably in cell proliferation and lineage determination. ELK-1 forms a ternary complex with a dimer of Serum Response Factor at Serum Response Elements associated with immediate-early genes, such as CFOS. Following mitogen-stimulated activation of the ERK cascade, ELK-1 is phosphorylated to mediate transcriptional activation of its target genes. Further to phosphorylation, ELK-1 is covalently modified post-translationally with both monoubiquitin and polyubiquitin chains. This study sought to understand the functional significance and regulation of ELK-1 ubiquitination. Sites of ubiquitination of ELK-1 ectopically expressed in HEK293T cells were mapped and screened for the signature diglycine motif associated with ubiquitinated tryptic peptides using liquid chromatography - tandem mass spectrometry (LC-MS/MS). This revealed that lysine residues within the amino-terminal DNA-binding domain of ELK-1 are monoubiquitinated, including those involved in DNA binding. Ubiquitination is reversed through the action of deubiquitinating enzymes (DUBs), which are target specific, allowing dynamic control over the protein modification state. USP17, one of several candidate DUBs, was shown to deubiquitinate ELK-1 when ectopically expressed in HEK293T cells. This was reversed when USP17 expression was knocked down, which also downregulated transcription of ELK-1 responsive genes CFOS and EGR1. Furthermore, USP17 knock-down in HEK293T cells reduced cell proliferation, an effect that was partially rescued by expression of a hypo-ubiquitinated ELK-1 mutant. Taken together, these results reveal monoubiquitination to be a key regulator of ELK-1 transcriptional potency and mitogen-driven proliferation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:748227 |
| Date | January 2018 |
| Creators | Ducker, Charles |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/48595/ |
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