Profiling deubiquitylase activity during the cell cycle reveals phosphorylation-dependent regulation of USP7 activity at G1/S

Darling, S (2017) Profiling deubiquitylase activity during the cell cycle reveals phosphorylation-dependent regulation of USP7 activity at G1/S. PhD thesis, University of Liverpool.

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Abstract

Ubiquitylation is a well-defined regulator of the cell cycle, as temporal degradation of cell cycle effectors is essential to ensure unidirectional progression. A family of approximately 100 deubiquitylases (DUBs) antagonise ubiquitylation and therefore may also play important roles in cell cycle progression. Several DUBs have been associated with the governance of checkpoint transitions through RNA interference screens, yet the periodic regulation of DUB catalytic activity remains unexplored. This project aimed to generate the first global study of cell cycle-dependent DUB regulation. Active site-directed ubiquitin probes were incubated with extracts from synchronised A549 cell populations enriched for key cell cycle phases. Those DUBs with accessible and reactive active sites were detected by specific immunoblotting, or unbiased triple-labelled mass spectrometry, to comprehensively profile global DUB activity throughout the cell cycle. Twenty-three DUBs were identified, most exhibited differential activity during the cell cycle and interestingly there was pervasive downregulation of DUB activity during mitosis. Further analysis revealed clusters of DUBs that were co-regulated during the cell cycle, the largest of which exhibited increased activity during S-phase coupled with a notable reduction in activity at mitosis. This periodic profile of DUB activity was observed for USP7. A comparative analysis of USP7 abundance and activity identified proportionally increased activity during G1/S; this coincided with an increase USP7 phosphorylation at serine 18 (S18). GFP-tagged USP7 constructs were generated harbouring non-phosphorylatable (S18A) or phospho-mimetic (S18E) mutations to investigate the role of phosphorylation in a cellular context. Exogenously expressed USP7-S18A, but not USP7-S18E, exhibited decreased reactivity towards active site-directed ubiquitin probes. Furthermore, both USP7-S18A and catalytically inactive USP7-C223S displayed increased cytoplasmic localisation compared to wild-type USP7. This suggests activity-dependent localisation that could alter the availability of USP7 for certain substrates. Importantly, the role of S18 phosphorylation was elucidated in an in vitro setting using bacterially expressed USP7. CK2 phosphorylation directly potentiated USP7 catalytic activity, increasing cleavage of K48-linked tetra-ubiquitin chains and ubiquitin-AMC compared to the non-phosphorylated USP7. Enzyme kinetics demonstrated 5-fold higher activity towards ubiquitin-AMC, predominantly through increased Kcat. This is the first report that S18-phosphorylation of USP7 fundamentally augments catalytic activity. In a cellular context, this is elevated at G1/S, affects subcellular compartmentalisation and may alter affinity towards specific substrates.

Item Type:	Thesis (PhD)
Divisions:	Faculty of Health and Life Sciences > Faculty of Health and Life Sciences
Depositing User:	Symplectic Admin
Date Deposited:	18 Sep 2017 13:55
Last Modified:	19 Jan 2023 07:02
DOI:	10.17638/03008142
Supervisors:	Coulson, J ORCID: 0000-0003-2191-2001
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3008142