Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy
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Thomas Mercer Yohei Ohashi Stefan Boeing Harold Jefferies Stefano De Tito Helen Flynn Shirley Tremel Wenxin Zhang Martina Wirth David Frith Bram Snijders Roger Lee Williams Sharon ToozeAbstract
Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK-dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK-dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation-independent accumulation of ULK substrates and kinase activity-regulated recruitment of autophagy proteins to ubiquitin-positive structures.
Journal details
Journal The EMBO Journal
Volume 40
Issue number 14
Pages e105985
Available online
Publication date
Full text links
Publisher website (DOI) 10.15252/embj.2020105985
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Europe PubMed Central 34121209
Pubmed 34121209