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Updated: Mar 6, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
Study of ULK1 Catalytic Activity and Its Regulation
1Institute of Molecular Medicine I, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany.
Abstract:
During the last decade, the molecular mechanisms controlling the initiation of (macro-)autophagy have been extensively studied. Two macromolecular kinase complexes are central for the initiation of autophagy: the protein kinase unc-51-like kinase 1 (ULK1) complex and the lipid kinase VPS34/Beclin 1 complex. The serine/threonine kinase ULK1 represents the mammalian ortholog of yeast autophagy-related (Atg) protein 1 (Atg1). ULK1 is regulated by upstream nutrient- and energy-sensing kinases, and transmits these signals to the core autophagic machinery. To date, the analysis of ULK1 activation and/or activity is an effective tool to investigate autophagy pathways. As described in this chapter, this can be performed by immunoblotting with phosphosite-specific antibodies against ULK1 and/or ULK1 substrates, by mass spectrometry, or by in vitro kinase assays. Furthermore, the recent design and development of ULK1-specific inhibitors established this kinase as an attractive therapeutic target in settings, where the inhibition of autophagy is desired.
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