Study of ULK1 Catalytic Activity and Its Regulation.

B Stork, J Dengjel

Methods in enzymology 2017

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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. © 2017 Elsevier Inc. All rights reserved.