Abstract
Autophagy is a crucial homeostatic mechanism that mediates the degradation of damaged or excess intracellular components. Such components are engulfed and sequestered into double membrane autophagosomes, which deliver their contents to lysosomes for degradation. Autophagy plays a role in numerous human disorders and its pharmacological targeting by small molecules offers therapeutic potential. The serine/threonine kinase ULK1 (and its homologue ULK2) is the most upstream component of the autophagic machinery and is required for autophagy initiation. Here, we use the most selective and potent published ULK1 inhibitors to gain insights into ULK1 kinase function during autophagy. Treatment with all inhibitors blocked autophagy but also resulted in the limited formation of initial autophagosome-like structures, which appeared abnormal in size and did not traffic to lysosomes. We found that upon ULK1 inhibition, phosphatidylinositol-3-phosphate-binding proteins are still recruited to forming autophagosomes, implying that ULK1 activity is not essential for VPS34 activation. We conclude that the kinase activity of ULK1 is important in regulating autophagosome maturation, by the phosphorylation of currently unidentified key substrates.
Highlights
Autophagy is a crucial homeostatic mechanism regulating lysosomedependent degradation and recycling of intracellular components
The GFP-LC3 signal appeared diffused in the cytoplasm but after 15 min of Earle’s balanced salt solution (EBSS) treatment GFP-LC3 formed numerous punctate structures, which continued to increase in number in a time-dependent manner (Fig 1A and B and Video 1). mCherry-DFCP1 appeared to already form punctate structures from the beginning, which could be representative of its known localisation on Golgi-derived membranes under basal conditions [16]
The presence of DFCP1, which requires PI3P for localisation, suggests that VPS34 is still active despite the fact that Unc-51 like autophagy-activating kinase 1 (ULK1) kinase activity is inhibited and is examined in more detail below
Summary
Autophagy is a crucial homeostatic mechanism regulating lysosomedependent degradation and recycling of intracellular components. The role of ULK1 during autophagy has been extensively studied in the context of amino acid starvation, where it has been shown (in multiple cell lines and tissues) that in a fed state, ULK1 is suppressed by direct Mechanistic target of rapamycin complex 1 (mTORC1)-mediated phosphorylation at multiple sites (including serine in murine or in human ULK1) [10]. PI3P production recruits PI3P-binding proteins such as WD-repeat protein interacting with Phosphoinositides (WIPI2) and Zinc Finger FYVE domain-containing protein 1 (DFCP1) [16, 17, 18] The role of these proteins is poorly understood, one known function of WIPI2 is to bind and recruit ATG16L1—an essential protein for autophagosome elongation and lipidation of the autophagosomal membrane microtubule associated protein 1 light chain 3 (MAP1LC3)—to the autophagosome forming sites [19].
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