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Post-translational modification in the regulation of the ubiquitin conjugating enzyme UBE2D3

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Title: Post-translational modification in the regulation of the ubiquitin conjugating enzyme UBE2D3
Authors: Ito, Constance Masako
Item Type: Thesis or dissertation
Abstract: UBE2D3 is a highly conserved and promiscuous E2 ubiquitin conjugating enzyme, which is known to function in processes that are important for normal development and dysregulated in cancers. This study sought to understand how the level of this protein is maintained and regulated by post-translational modifications in mouse embryonic stem cells (mESCs) and non-small cell lung carcinoma (NSCLC) cells. Conserved residues in the C-terminal α-helix and active site of UBE2D3, were found to be essential in the regulation of the protein stability in mESCs. These residues included C85, S94, T142, S138 and K144, whose mutation indirectly implicated phosphorylation, ubiquitination and nitrosylation, alongside possible mechanisms of protein interactions and direct structural effects. UBE2D3-S138 has been shown to be the site of phosphorylation by Aurora B kinase. This thesis presents evidence that phosphorylation of UBE2D3-S138 by Aurora B kinase in vitro, intrinsically disrupts the protein structure causing insolubility. Expression of phospho-mimetic mutants of UBE2D3 in mESCs confirmed the destabilising effect of S138 phosphorylation. This mechanism is essential for mouse embryonic development, and it has been shown to be required during differentiation into primitive endoderm, but was not critical in the formation of the germ layer lineages in this thesis. The stability of UBE2D3 is also dependent on the invariant K144. K144 is known to be ubiquitinated in vivo and when it was mutated to arginine in mESCs, UBE2D3 was downregulated and the mutant cells were incapable of effective differentiation into primitive endoderm and the lineages of the three germ layers. We conclude that the post-translational regulation of UBE2D3 protein levels, by conserved residues and their modification, is critical for the pluripotency of mESCs. The investigation of UBE2D3 in NSCLC cells revealed a similar mechanism of UBE2D3 destabilisation by Aurora B kinase, and a role for UBE2D3 activity in cell survival and proliferation.
Content Version: Open Access
Issue Date: Aug-2019
Date Awarded: Feb-2020
URI: http://hdl.handle.net/10044/1/92936
DOI: https://doi.org/10.25560/92936
Copyright Statement: Creative Commons Attribution NonCommercial NoDerivatives Licence
Supervisor: Dillon, Niall
Sponsor/Funder: Medical Research Council (Great Britain)
Department: Institute of Clinical Sciences
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Department of Clinical Sciences PhD Theses



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