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Investigating the regulation of WIPI2b function at the phagophore by phosphorylation in starvation-induced autophagy

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Title: Investigating the regulation of WIPI2b function at the phagophore by phosphorylation in starvation-induced autophagy
Authors: Gubas, Andrea
Item Type: Thesis or dissertation
Abstract: Macroautophagy, here referred to as autophagy, is an intracellular degradation pathway cells use to maintain their homeostasis. Autophagy is also required for cell survival during nutrient deprivation, as well as development and immunity in higher eukaryotes. Aberrations in autophagy can lead to pathologies including cancer, neurodegeneration and diabetes. Autophagy is characterised by the formation of a double membrane phagophore, which sequesters cytosolic cargo and forms a vesicle termed an autophagosome. The autophagosome eventually fuses with the lysosome, resulting in the degradation of the cytosolic cargo. Although autophagosome formation is orchestrated by the sequential action of the core autophagy proteins, a key question remains – what gives rise to a double membrane phagophore? The key event in phagophore biogenesis is the production of PI3P at the phagophore formation sites. WIPI2b, a PI3P effector protein, directly interacts with ATG16L1 and is recruited to the omegasomes, which is the basis for LC3 recruitment to the forming phagophore. To address how the function of WIPI2b at the phagophore is regulated, I focused on phosphorylation, as there have been reports about potential phosphorylation sites on WIPI2b. I confirmed an interactive relationship between WIPI2b and ULK1 that was reported previously and identified a number of phosphorylation sites on WIPI2b upon overexpression of ULK1 kinase. I found that phospho-mutants of WIPI2b S68 exhibit reduced interaction with ATG16L1 and WIPI4. I generated and characterised a WIPI2 CRISPR knockout cell line and found that WIPI2b S68 phospho-mutants are unable to rescue LC3 lipidation in WIPI2 CRISPR knockout cells. I also found that WIPI2b S284 phosphorylation is important for the regulation of WIPI2b association with membranes. I propose WIPI2b phosphorylation by ULK1 provides a feedback loop during autophagy to control the amount of functional WIPI2b at phagophores and therefore allows phagophore elongation.
Content Version: Open Access
Issue Date: Sep-2018
Date Awarded: Dec-2018
URI: http://hdl.handle.net/10044/1/75543
DOI: https://doi.org/10.25560/75543
Copyright Statement: Creative Commons Attribution NonCommercial NoDerivatives Licence
Supervisor: Tooze, Sharon
Braga, Vania
Department: Francis Crick Institute
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses