Abstract
Autophagy is a catabolic cellular mechanism for entrapping cellular macromolecules and organelles in intracellular vesicles and degrading their contents by fusion with lysosomes. Important roles for autophagy have been elucidated for cell survival during nutrient insufficiency, eradication of intracellular pathogens, and counteracting aging through clearance of senescent proteins and mitochondria. Autophagic vesicles become decorated with LC3, a protein that mediates their fusion with lysosomes. LC3 is a substrate of the cysteine protease ATG4B (Autophagin-1), where cleavage generates a C-terminal glycine required for LC3 conjugation to lipids in autophagosomes. ATG4B both cleaves pro-LC3 and also hydrolyzes lipids from cleaved LC3. We show here that phosphorylation of ATG4B at Ser-383 and Ser-392 increases its hydrolyase activity as measured using LC3 as a substrate. Reconstituting atg4b(-/-) cells with phosphorylation-deficient ATG4B showed a role of ATG4B phosphorylation in LC3 delipidation and autophagic flux, thus demonstrating that the cellular activity of ATG4B is modulated by phosphorylation. Proteolytic conversion of pro-LC3 to LC3-I was not significantly impacted by ATG4B phosphorylation in cells. Phosphorylation-deficient ATG4B also showed reduced interactions with the lipid-conjugated LC3 but not unconjugated LC3. Taken together, these findings demonstrate a role for Ser-383 and Ser-392 phosphorylation of ATG4B in control of autophagy.
Highlights
ATG4B mediates the cleavage of pro-light chain 3 (LC3) and removes lipid conjugates from LC3 during autophagy
ATG4B Is Phosphorylated in Cells—To determine whether the cysteine protease ATG4B is phosphorylated in cells, we expressed functional ATG4B tagged with Flag peptide in HEK293T cells
Our data suggest that phosphorylation of residues located in the C terminus of ATG4B plays a role in promoting autophagy, enhancing the ability of ATG4B to delipidate its endogenous substrate LC3
Summary
ATG4B mediates the cleavage of pro-LC3 and removes lipid conjugates from LC3 during autophagy. Phosphorylation-deficient ATG4B showed reduced interactions with the lipid-conjugated LC3 but not unconjugated LC3 Taken together, these findings demonstrate a role for Ser-383 and Ser-392 phosphorylation of ATG4B in control of autophagy. ATG4B overexpression resulted in an increase of LC3-I, which was attributed to LC3-II deconjugation [7], whereas upregulating ATG4B by aplasia Ras homolog member I (ARH1) or stabilizing ATG4B protein levels by inactivation of the E3 ligase RNF5 was associated with increased LC3 processing [19, 20] These observations indicate a convoluted and critical role for ATG4B in the mechanisms that control autophagy. Further analyses revealed that phosphorylation of ATG4B regulates LC3 lipidation in cells, and thereby modulates autophagic flux
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