eprintid: 10086328
rev_number: 28
eprint_status: archive
userid: 608
dir: disk0/10/08/63/28
datestamp: 2019-12-13 09:58:21
lastmod: 2020-12-01 07:10:46
status_changed: 2019-12-13 09:58:21
type: thesis
metadata_visibility: show
creators_name: Mercer, Thomas John
title: Examining the mechanistic regulation of starvation-induced autophagy via the identification and characterisation of novel ULK kinase substrates
ispublished: unpub
divisions: UCL
divisions: A01
divisions: B02
divisions: C08
note: Copyright © The Author 2019.  Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/).  Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms.  Access may initially be restricted at the author’s request.
abstract: Autophagy involves the formation of an endoplasmic reticulum-derived membrane termed a phagophore which expands to engulf cytoplasmic cargo before sealing to form an autophagosome. Amino acid starvation is amongst the most potent autophagic stimuli, however whilst the key signalling complexes involved in starvation-induced autophagy are known, the precise regulatory mechanisms remain poorly understood. The serine/threonine kinase ULK1 and close homolog ULK2 assume the most upstream position in the autophagic signalling cascade and play a crucial yet enigmatic role in coordinating the autophagic machinery. To further understand the mechanisms of starvation-induced autophagy, I performed a number of unbiased phosphoproteomic screens to identify ULK substrates before classifying their roles in starvation-induced autophagy. Analysis of these datasets has revealed that loss of ULK results in significant changes to the phosphoproteome and has yielded a high confidence list of potential substrates whilst also offering interesting insights into the veracity of the published ULK consensus signature. Amongst the novel phosphorylation targets are components of the retromer and AMPK complexes along with multiple components of the class III PI3K VPS34 complex. The pseudokinase p150, scaffolding component of the VPS34 complex, is phosphorylated by ULK1 in vitro and in vivo at serine 861. CRISPR-based knockout of p150 results in inhibition of autophagy and endosomal trafficking, whilst mutating the phosphorylated residue in p150 alters both omegasome establishment and autophagic flux. Furthermore, incorporation of phosphomutant p150 into the VPS34 complex modulates its lipid kinase activity in vitro. These data identify a novel ULK-dependent signalling axis and help illuminate the complexities of signal transduction in autophagy.
date: 2020-11-28
date_type: published
oa_status: green
full_text_type: other
thesis_class: doctoral_open
thesis_award: Ph.D
language: eng
thesis_view: UCL_Thesis
primo: open
primo_central: open_green
verified: verified_manual
elements_id: 1720414
lyricists_name: Mercer, Thomas
lyricists_id: TMERC33
actors_name: Mercer, Thomas
actors_name: Allington-Smith, Dominic
actors_id: TMERC33
actors_id: DAALL44
actors_role: owner
actors_role: impersonator
full_text_status: public
pagerange: 1-311
pages: 311
event_title: UCL (University College London)
institution: UCL (University College London)
department: MCBA Lab- The Francis Crick Institute
thesis_type: Doctoral
editors_name: Tooze, S
citation:        Mercer, Thomas John;      (2020)    Examining the mechanistic regulation of starvation-induced autophagy via the identification and characterisation of novel ULK kinase substrates.                   Doctoral thesis  (Ph.D), UCL (University College London).     Green open access   
 
document_url: https://discovery.ucl.ac.uk/id/eprint/10086328/1/Thesis%20%28Corrections%20Complete%29.pdf