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Non-canonical PKCε activation is required for the cellular response to TopoIIα inhibition

Kelly, Joanna; (2018) Non-canonical PKCε activation is required for the cellular response to TopoIIα inhibition. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The cell cycle is tightly regulated to safeguard the complete and equal division of the nuclear contents between the daughter cells. To ensure accurate segregation of the chromatin, DNA catenation generated during replication must be resolved by topoisomerase IIα (TopoIIα). In some tumour-derived cells, Protein kinase C epsilon (PKCε) has been shown to play a crucial role in mediating a delay signal at the metaphase-to-anaphase transition when catenation is still present, allowing time for catenation resolution prior to anaphase entry and sister chromatid separation. PKCε has also been shown to affect the decatenation process itself, with an increase in metaphase catenation being observed upon PKCε knock down or inhibition. Active PKCε is predominantly membrane associated as lipid binding is required for canonical activation. However, a lipid-independent activation pathway has been observed which involves complex formation with the scaffold protein 14-3-3. In this thesis, we have identified a second lipid-independent pathway. In mitosis, we have identified a caspase-mediated, proteolytic cleavage of PKCε within a chromatin-associated sub-compartment. The cleavage occurs at two distinct sites, one of which leads to the generation of a free kinase domain. Blocking PKCε cleavage at this site, via mutation or caspase-7 inhibition, results in a reduction in the PKCε dependent delay to the metaphase-to-anaphase transition. This can be rescued by artificially recapitulating the cleavage event. Cells expressing a non-cleavable PKCε also retain larger amounts of metaphase catenation, indicating that cleavage is required to support resolution as well as the delay. As both of these phenotypic responses require active PKCε this proteolytic cleavage represents a novel, non-canonical activation pathway. The mechanism by which the free kinase domain generated influences the response to TopoIIα inhibition is still to be fully resolved but phosphorylation of the key mitotic regulator Aurora B and subsequent phosphorylation of TopoIIα have been shown to play a crucial role. Due to an increased dependence on this response in a number of tumour-derived cells, identifying the pathways involved in its regulation may offer a potentially novel target for cancer therapeutics.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Non-canonical PKCε activation is required for the cellular response to TopoIIα inhibition
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10045030
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