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. 2017 Aug 18;8(1):294.
doi: 10.1038/s41467-017-00303-2.

A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B

N Pengo  1 , A Agrotis  1 , K Prak  1 , J Jones  1 , R Ketteler  2
Affiliations

A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B

N Pengo et al. Nat Commun. .

Abstract

Upon induction of autophagy, the ubiquitin-like protein LC3 is conjugated to phosphatidylethanolamine (PE) on the inner and outer membrane of autophagosomes to allow cargo selection and autophagosome formation. LC3 undergoes two processing steps, the proteolytic cleavage of pro-LC3 and the de-lipidation of LC3-PE from autophagosomes, both executed by the same cysteine protease ATG4. How ATG4 activity is regulated to co-ordinate these events is currently unknown. Here we find that ULK1, a protein kinase activated at the autophagosome formation site, phosphorylates human ATG4B on serine 316. Phosphorylation at this residue results in inhibition of its catalytic activity in vitro and in vivo. On the other hand, phosphatase PP2A-PP2R3B can remove this inhibitory phosphorylation. We propose that the opposing activities of ULK1-mediated phosphorylation and PP2A-mediated dephosphorylation provide a phospho-switch that regulates the cellular activity of ATG4B to control LC3 processing.Upon autophagy induction, LC3 is cleaved by the protease ATG4 and conjugated to the autophagosomal membrane; however, its removal is mediated by the same protease. Here the authors show that ULK1-mediated phosphorylation and PP2A-mediated dephosphorylation of ATG4 regulates its cellular activity to control LC3 processing.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
ULK1 inhibits ATG4B-mediated LC3 cleavage. a Average ATG4B activity in Actin-LC3-DelN Luciferase HEK293T obtained by measuring the secreted luciferase activity 48 h after transfection with the indicated constructs (n = 3, average ± s.d.) and representative immunoblot from one experiment showing expression of the different constructs. b GST-LC3 assay to measure in vitro activity of recombinant ATG4B after incubation with active recombinant ULK1. Representative gel and quantification of free cleaved LC3 (n = 3, average ± s.d.). c GST-LC3 assay to measure in vitro activity of recombinant ATG4B after incubation with active recombinant ULK1 in the presence or absence of 1 mM ATP. Representative gel showing three independent replicates for each condition and d quantification of free cleaved LC3 (n = 3, average ± s.d.)
Fig. 2
Fig. 2
ATG4B Ser316 is a target residue of ULK1 phosphorylation. a Clustal Omega multiple alignment of ATG4 protein sequences surrounding hATG4B serine 316 from different isoforms and species; highlighted in green are serine residues and in violet hydrophobic amino acids. b In vitro radioactive phosphorylation assay with WT or ATG4B S316A mutant at different time points after addition of recombinant active ULK1. Coomassie blue staining is shown as an ATG4B loading control. c GST-ATG4B or GST-ATG4B S316A mutant was incubated for the indicated times with GST-ULK1 (1–283); samples were then assayed with a custom phospho-specific antibody against Ser316 of human ATG4B—pATG4B (Ser316) or total ATG4B. d Total lysates from wild-type (WT) or ULK1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs) transfected with 3×-FLAG-tagged ATG4B or mCherry as a control and treated with 1 µM okadaic acid for 1 h were probed with the pATG4B(Ser316) antibody and with an anti-ACTIN antibody as loading control. In parallel, lysates from the same samples were subjected to immunoprecipitation with FLAG M2 affinity gel and probed for pATG4B(Ser316) and FLAG antibody. Phosphorylation level was calculated using densitometry, with pATG4B(Ser316) signal divided by FLAG signal for the same band, expressed as a percentage of the WT MEF IP sample. e HEK293T lysates from samples co-transfected with mouse WT myc-ULK1 or Kinase Inactive (KI) myc-ULK1 and the indicated Halo constructs for 24 h were blotted and probed with the indicated antibodies (one representative blot from three independent experiments)
Fig. 3
Fig. 3
ATG4B Ser316 phosphorylation reduces LC3 binding and inhibits its catalytic activity towards unprocessed and lipidated LC3 in vitro and in cells. a Overview of the ATG4B-LC3 co-crystal structure from PDB 2Z0E, zooming at ATG4B Ser316 location. LC3 is in yellow and ATG4B is in green. Red and blue represent the oxygen and nitrogen atoms, respectively. Hydrogen bond is shown as a black dashed line. The molecular structure was generated with PyMOL. b HeLa cells stably expressing GFP-tagged LC3B were transfected with 3×FLAG-tagged ATG4B and ATG4B mutants or mCherry as control and subjected to immunoprecipitation with FLAG M2 affinity gel. The blots were probed for interaction of the 3×FLAG-tagged proteins with GFP-LC3B using GFP antibody (upper panel) and endogenous LC3B with LC3B antibody (lower panel), and 3×FLAG-ATG4B was detected using FLAG antibody (middle panel). c ATG4B activity was measured in the Actin-LC3-DelN reporter cell line 48 h after transfection with the WT, catalytic inactive (C74S), phosphomimetic (S316D) and non-phosphorylatable (S316A) Halo-ATG4B constructs (n = 3, average ± s.d., and representative blot showing the correct expression of the constructs). Values displayed are the relative intensity of phosphorylated ATG4B Ser316 normalized to Halo-ATG4B, relative to WT HaloATG4B expression (unpaired two-sided t-test). d Purified recombinant WT, phosphomimetic (S316D) and catalytic inactive (C74S) GST-ATG4B (0.004 mg mL−1) were tested in vitro for their ability to cleave LC3B-GST (1 mg mL−1). e Cytoplasmic and non-nuclear membrane fraction preparations from HEK293T cells treated with 250 nM Torin and 10 nM bafilomycin A1 for 3 h were incubated with the indicated GST-ATG4B WT, S316D and C74S mutants for 30 min at 37 °C, blotted and probed with the indicated antibodies. LAMP1 and PRDX1 were used as loading controls for the membrane and cytoplasmic fractions, respectively, and GST-ATG4B was detected using anti-ATG4B (one representative blot is shown)
Fig. 4
Fig. 4
Quantification of LC3-positive autophagosomes in ATG4B knockout cells rescued with the phosphomimetic ATG4B S316D mutant. a Validation of CRISPR-mediated modification to the genomic locus of ATG4B exon 4 in the HAP1 ATG4B KO cell line. The gene structure of the WT human ATG4B locus on chromosome 2 is shown, and the region in exon 4 targeted for modification is revealed at the sequence level. The DNA sequence targeted by the custom sgRNA is shown in bold. The 16 bp deletion in the ATG4B KO clone results in a frameshift and early stop codon, as shown by the DNA sequence alignment. b Protein-level validation of ATG4B knockout. Lysates from untreated HAP1 control and ATG4B KO cells were run on a 4–20% polyacrylamide gel and immunoblotted with antibodies against ATG4B and beta-actin. The ATG4B KO cells showed a total absence of the specific band representing ATG4B. c ATG4B KO HAP1 cells were transfected with the indicated Halo constructs. After 24 h, cells were simultaneously stained live with a Halo ligand dye and treated with 250 nM Torin1 and 10 nM bafilomycin A1 for 3 h, prior to fixation and staining for endogenous LC3. Arrowheads indicate high Halo construct-expressing cells, and arrows indicate cells expressing low levels of these constructs. Scale bar 10 µm. d Quantification of the average number of autophagosome per cell in the high expressing cells is shown (Tukey’s plot, n = 10 fields, with an average of 5 quantified cells per field). ****p < 0.0001, NS not significant (Sidak’s multiple comparison test)
Fig. 5
Fig. 5
A phosphatase screen identifies PP2A as an endogenous regulator of ATG4B Ser316 dephosphorylation. a Schematic workflow of the cDNA phosphatase screen. b HEK293T cells after 24 h co-transfection with selected phosphatases and myc-ULK1 and Halo-ATG4B were analysed for the phosphorylation status of ATG4B on Ser316. The samples overexpressing the PP2A regulatory subunit 3B (PPP2R3B) are marked with an asterisk. Please note that the three different cDNA clones were transfected for PPP2R3B, all of which showed a reduction in phosphor-Ser316 compared with the control sample. Representative immunoblots are shown. c Cell lysates from HeLa cells stably expressing Halo-ATG4B WT and treated for 30 min with 1 µM okadaic acid or DMSO were probed for phosphorylation of ATG4B on Ser316 and then re-probed for Halo and β-actin expression. Representative immunoblot from three independent experiments
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