title: ULK1-mediated phosphorylation of ATG14 promotes autophagy and is impaired in Huntington's disease models.
abstract: Autophagy is a bulk degradation pathway for long-lived proteins, protein aggregates, and damaged organelles. ULK1 protein kinase and Vps34 lipid kinase are two key autophagy regulators that are critical for autophagosome biogenesis. However, it isn't fully understood how ULK1 regulates Vps34, especially in the context of disease. Polyglutamine expansion in huntingtin (Htt) causes aberrant accumulation of the aggregated protein and disrupts various cellular pathways including autophagy, a lysosomal degradation pathway, underlying the pathogenesis of Huntington's disease (HD). Although autophagic clearance of Htt aggregates is under investigation as therapeutic strategy for HD, the precise mechanism of autophagy impairment remains poorly understood. Moreover, in-vivo assays of autophagy have been particularly challenging due to lack of reliable and robust molecular biomarkers.
  
  
  
  We generated anti-phosphorylated ATG14 antibody to determine ATG14-mediated autophagy regulation; we employed Huntington's disease (HD) genetic cell models and animal models as well as autophagy reporter animal model to understand autophagy signaling and regulation in vivo. We applied biochemical analysis and molecular biology approaches to dissect the alteration of autophagy kinase activity and regulation.
  
  
  
  Here, we demonstrate that ULK1 phosphorylates ATG14 at serine 29 in an mTOR-dependent manner. This phosphorylation critically regulates ATG14-Vps34 lipid kinase activity to control autophagy level. We also show that ATG14-associated Vps34 activity and ULK1-mediated phosphorylation of ATG14 and Beclin 1 are compromised in the Q175 mouse model of Huntington's disease. Finally, we show that ATG14 phosphorylation is decreased during general proteotoxic stress caused by proteasomal inhibition. This reduction of the specific phosphorylation of ATG14 and Beclin 1 is mediated, in part, by p62-induced sequestration of ULK1 to an insoluble cellular fraction. We show that increased ULK1 levels and phosphor-mimetic mutant ATG14 facilitate the clearance of polyQ mutant in cells.
  
  
  
  Our study identifies a new regulatory mechanism for ATG14-Vps34 kinase activity by ULK1, which can be used as valuable molecular markers for in-vivo autophagic activity as well as potential therapeutic target for the clearance of polyglutamine disease protein.
authors.affiliation: Department of Neurology, The Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
authors.forename: Mitchell S
authors.initials: MS
authors.lastname: Wold
authors.affiliation: Department of Neurology, The Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.|Present Address: Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080, USA.
authors.forename: Junghyun
authors.initials: J
authors.lastname: Lim
authors.affiliation: Department of Neurology, The Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
authors.forename: Véronik
authors.initials: V
authors.lastname: Lachance
authors.affiliation: Department of Neurology, The Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.|Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, and Medical Research Institute, Wuhan University, Wuhan, 430071, China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Deng
authors.affiliation: Department of Neurology, The Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. zhenyu.yue@mssm.edu.|Leon and Norma Hess Center for Science and Medicine, 9-106, 1470 Madison Ave, New York, NY, 10029, USA. zhenyu.yue@mssm.edu.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Yue
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000818:Animals; D001343:Autophagy / Q000502:physiology*; D000071189:Autophagy-Related Protein-1 Homolog / Q000378:metabolism*; D000071183:Autophagy-Related Proteins / Q000378:metabolism*; D002460:Cell Line; D004195:Disease Models, Animal; D006801:Humans; D006816:Huntington Disease / Q000378:metabolism*; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D008856:Microscopy, Fluorescence; D010766:Phosphorylation
chemical_list: C539000:ATG14 protein, human; D033942:Adaptor Proteins, Vesicular Transport; D000071183:Autophagy-Related Proteins; D000071189:Autophagy-Related Protein-1 Homolog
keywords: ATG14; Autophagy; Huntington’s; ULK1; Vps34