Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells

Abstract

Autophagy protease ATG4B is a key regulator of the LC3/GABARAP conjugation system required for autophagosome formation, maturation and closure. Members of the ATG4 and the LC3/GABARAP family have been implicated in various diseases including cancer, and targeting the ATG4B protease has been suggested as a potential therapeutic anti-cancer strategy. Recently, it has been demonstrated that ATG4B is regulated by multiple post-translational modifications, including phosphorylation and de-phosphorylation. In order to identify regulators of ATG4B activity, we optimized a cell-based luciferase assay based on ATG4B-dependent release of Gaussia luciferase. We applied this assay in a proof-of-concept small molecule compound screen and identified activating compounds that increase cellular ATG4B activity. Next, we performed a high-throughput screen to identify kinases and phosphatases that regulate cellular ATG4B activity using siRNA mediated knockdown and cDNA overexpression. Of these, we provide preliminary evidence that the kinase AKT2 enhances ATG4B activity in cells. We provide all raw and processed data from the screens as a resource for further analysis. Overall, our findings provide novel insights into the regulation of ATG4B and highlight the importance of post-translational modifications of ATG4B.

On ATG4B as Drug Target for Treatment of Solid Tumours—The Knowns and the Unknowns

“…This inhibitory loop is displaced upon binding to LC3/GABARAP and, in addition, is most likely subject to conformational change induced by post-translational modification. For instance, Ser34, which is located at the hinge of the N-terminal domain, has been shown to be phosphorylated by AKT kinase [14], and a phospho-mimetic S34D mutant displays enhanced ATG4B activity [72], suggesting that phosphorylation results in a conformational opening of the auto-inhibitory loop.…”

“…The mechanism of GLUC release in this system is not well understood, but it does not require autophagy as it is not affected in ATG5 knockout cells [111], making this a specific ATG4B activity assay. This assay has been employed to identify regulators of ATG4B activity and has become one of the main methods to monitor cell-based ATG4 activity [12,59,72]. The assay has also been adapted to monitor LC3A and LC3B2 [9], thus broadening its application to other ATG4 family members, although a similar approach to monitor GABARAP cleavage instead of LC3 resulted in very high background release of GLUC, and is thus impractical for use as a phenotypic reporter for screening (R.K., unpublished observation).…”

“…This assay has been used to validate ATG4B inhibitors in cells, but the dynamic range of this assay is limited due to a high background activity. An assay to monitor transcriptional activation of ATG4B by fusion of the ATG4B promoter with renilla luciferase has also been reported [72], which may be useful for investigating upstream regulators of ATG4B expression. For instance, this assay has been used to identify small molecule compounds that activate ATG4B expression [72].…”

A new patient‐derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α‐dystroglycan

“…Large-scale screening was performed on 31,954 chemical compounds from the ChemiBank(UCL) collection (http://www.ucl.ac.uk/chemibank/) [66]. The flasks were coated with 0.1 mg/ml Matrigel (BD bioscience) diluted in DMEM (Gibco, Life Technology).…”

“…The growth medium consists of 20% fetal bovine serum (FBS, Gibco), 2% L-glutamine (Sigma), and 1% penicillin/streptomycin (Gibco), plus 2% chick embryo extract (Sera lab) and c-IFN (2 ll/ml, Merck). Large-scale screening was performed on 31,954 chemical compounds from the ChemiBank(UCL) collection (http://www.ucl.ac.uk/chemibank/) [66]. Molecular properties ranges were as follows: molecular weight between 126 and 600, AlogP between À3.5 and 6, hydrogen bond donors between 0 and 6, hydrogen bond acceptors between 0 and 12, rotatable bonds between 0 and 15, and number of rings between 1 and 8.…”