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Title: Investigating Polo-like kinase 4-regulated signalling using SILAC-based quantitative phosphoproteomics
Author: Ferries, Samantha
ISNI:       0000 0004 7656 8815
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Protein phosphorylation plays a critical role in regulating cellular responses, including the coordination of cell division. Polo-like kinase 4 (PLK4), a cell cycle regulated Ser/Thr protein kinase, is the master regulator of centriole biogenesis, a process required for formation of the biopolar mitotic spindle. PLK4 expression and activity are both very tightly controlled, and dysregulation leads to aberrant centrosome duplication resulting in chromosome missegregation and aneuploidy. Despite its rising importance as a potential anti-cancer target, few PLK4 substrates have been identified, and much remains to be understood about basic PLK4 biology at the centrosome, and wider potential roles within the cell. The experiments described in this thesis focused on an evaluation of PLK4 signalling, exploiting SILAC-based quantitative phosphoproteomics and the protein kinase inhibitor centrinone to probe cellular function. Initially, I describe the development of biochemical tools to study PLK4-dependent signalling, and its inhibition by centrinone. This included the generation of stable isogenic U2OS cell lines, transfected with either WT PLK4 or a centrinone-resistant G95R PLK4 mutant. The G95R PLK4 cell line was designed to explore potential off-target effects of centrinone inhibition, and biochemical and cellular assays confirmed the 'drug-resistant' status of this protein. An optimal concentration of centrinone was established to ensure inhibition of WT PLK4, whilst minimising off-target effects. Next, mass spectrometric (MS) acquisition parameters for phosphopeptide analysis were optimised on an Orbitrap Fusion, using a synthetic phosphopeptide library and phosphopeptide-enriched cell lysate. Eight acquisition methods were assessed, considering mode of fragmentation (HCD, EThcD, and neutral loss triggered ET(ca/hc)D), and analysers for MS2 (orbitrap and ion trap) in addition to assessment of optimal downstream processing. HCD, with orbitrap MS2 analysis provided high numbers of identifications with high phosphosite localisation and was used to study the PLK4 regulated phosphoproteome. Building upon this, phosphoproteomics subsequently revealed 412 significantly (p =0.075) regulated proteins in WT PLK4 expressing cells following centrinone treatment, and 471 regulated phosphosites, with 313 downregulated (inhibited) and 158 upregulated (activated) after compound exposure. A target of centrinone was implicated in MAPK signalling and G1/S phases of the cell cycle. In addition, regulation of biological processes including rRNA processing, cell-adhesion and transcription was observed. A novel PLK4 proline directed consensus phosphorylation motif was also identified from phosphoproteomic data and validated biochemically with recombinant PLK4 in vitro. Finally, PLK4 was demonstrated to be a biochemical upstream activator of Aurora A kinase, phosphorylating it at Thr288 within the activation loop, which led to hyperactivation. In addition, both proteins co-localised in human cultured interphase cells, suggesting potential novel functions for PLK4-regulated Aurora A in the early stages of the cell cycle. Taken together, this work provides a series of novel insights into PLK4-regulated signalling, which will be useful for future studies evaluating the biological and disease-related functions of this important fundamental protein kinase.
Supervisor: Eyers, Claire ; Eyers, Patrick ; Beynon, Robert Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral