AbstractMacroautophagy (hereby autophagy) is a catabolic pathway whereby a double membrane organelle called an autophagosome, engulfs cytoplasmic components in order to degrade them via the lysosome. While autophagy is induced in response to various stresses to promote homeostasis and cell survival, a key mechanistic step in initiation of autophagosome formation is thought to be regulated by the serine/threonine kinases ULK1/2. ULK1/2 kinase activity is required for amino acid starvation-induced and other types of autophagy. Our lab recently showed that inhibition of ULK1 with the potent compound MRT68921, results in formation of stalled autophagosomes that are positive for early and late autophagy markers. These data suggested that ULK1 is not only regulating initiation of autophagosome formation but also later stages, such as elongation and fusion with lysosomes. Here, I compared the most selective and potent ULK1 inhibitors available to date as to what autophagosomal defects they cause. From this work it is concluded that ULK1 inhibition may block autophagosome flux by affecting dissociation of autophagy-initiating protein complexes from the forming autophagosome and influencing its closure. In order to understand the underlying mechanisms regulating this process I performed a proximity ligation assay (BioID) using MRT68921 as a tool to enrich for autophagy regulators trapped on stalled autophagosomes. With this approach ATG2B was identified, among others, as a protein that is enriched on autophagosomal structures upon ULK1 inhibition. I here show that ULK1 phosphorylates ATG2B upon autophagy induction in response to amino acid starvation. It is known that loss of ATG2A/B results in accumulation of arrested autophagosomes, consistent with that seen by ULK1 kinase inhibition. Thus, ATG2B and its homologue ATG2A might be key substrates of ULK1, the phosphorylation of which is required for proper autophagosome formation.
|Date of Award||2020|
|Supervisor||Ian Ganley (Supervisor)|
Investigating the role of ULK1 kinase activity during autophagy
Zachari, M. (Author). 2020
Student thesis: Doctoral Thesis › Doctor of Philosophy