Kinase-Inactivated ULK Proteins Inhibit Autophagy via Their Conserved C-Terminal Domains Using an Atg13-Independent Mechanism

Secretory Pathways Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
Molecular and Cellular Biology (Impact Factor: 4.78). 11/2008; 29(1):157-71. DOI: 10.1128/MCB.01082-08
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The yeast Atg1 serine/threonine protein kinase and its mammalian homologs ULK1 and ULK2 play critical roles during the activation
of autophagy. Previous studies have demonstrated that the conserved C-terminal domain (CTD) of ULK1 controls the regulatory
function and localization of the protein. Here, we explored the role of kinase activity and intramolecular interactions to
further understand ULK function. We demonstrate that the dominant-negative activity of kinase-dead mutants requires a 7-residue
motif within the CTD. Our data lead to a model in which the functions of ULK1 and ULK2 are controlled by autophosphorylation
and conformational changes involving exposure of the CTD. Additional mapping indicates that the CTD contains other distinct
regions that direct membrane association and interaction with the putative human homologue of Atg13, which we have here characterized.
Atg13 is required for autophagy and Atg9 trafficking during autophagy. However, Atg13 does not bind the 7-residue dominant-negative
motif in the CTD of ULK proteins nor is the inhibitory activity of the CTDs rescued by Atg13 ectopic expression, suggesting
that in mammalian cells, the CTD may interact with additional autophagy proteins.

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Article: Kinase-Inactivated ULK Proteins Inhibit Autophagy via Their Conserved C-Terminal Domains Using an Atg13-Independent Mechanism

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    ABSTRACT: Autophagy is a degradation pathway involving lysosomal machinery for degradation of damaged organelles like endoplasmic reticulum, mitochondria etc. into their building blocks to maintain homeostasis within the cell. ULK1, a serine/threonine kinase, is conserved across species, from yeasts to mammals, and plays a central role in autophagy pathway. It receives signals from upstream modulators such as TIP60, mTOR and AMPK and relays them to its downstream substrates like Ambra1 and ZIP kinase. The activity of this complex is regulated through protein-protein interactions and post-translational modifications. Applying in silico analysis we identified (i) conserved patterns of ULK1 that showed its evolutionary relationship between the species which were closely related in a family compared to others. (ii) total 23 TFBS distributed throughout ULK1 and nuclear factor (erythroid-derived) 2 (NFE2) is of utmost significance because of its high importance rate. NEF2 has already been shown experimentally to play role autophagy pathway. Most of these were of Zinc coordinating class and we suggest that this information could be utilized to modulate this pathway by modifying interactions of these TFs with ULK1. (iii) CATTT haplotype was prominently found with frequency 0.774 in the studied population and nsSNPs which could have harmful effect on ULK1 protein and these could further be tested. (iv) total 83 phosphorylation sites were identified; 26 are already known and 57 are new that include one at tyrosine residue which could further be studied for its involvement in ULK1 regulation and hence autophagy. Furthermore, 4 palmitoylation sites at positions 426, 927, 1003 and 1049 were also found which could further be studied for protein-protein interactions as well as in trafficking. Copyright © 2015. Published by Elsevier B.V.
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    • "The complexity of the regulation of autophagy by MTOR is illustrated by the fact that MTOR not only inhibits ULK1 but that , conversely , ULK1 also inhibits MTOR by phos - phorylation . This inhibition of MTORC1 by ULK1 may serve to amplify and stabilize initially small changes in nutrient signaling ( Chang et al . 2009 ; Jung et al . 2011 ) . Autophagy is also controlled by PKB . Short - term regu - lation occurs by PKB - dependent phosphorylation of Beclin1 ( Wang et al . 2012 ) ( Fig . 2 ) . Long - term regulation by PKB occurs by phosphorylation of FoxO3 , another transcription factor responsible for the synthesis of ATG proteins ( Mammucari et al "
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    • "The following primary antibodies were used for western blotting: Polyclonal anti-ULK1 (Santa Cruz sc-33182), monoclonal anti-TYR (Santa Cruz sc-20035), polyclonal TYRP1 (Santa Cruz sc-25543), polyclonal tubulin (Abcam ab6046), monoclonal Actin (Abcam ab11003), polyclonal anti-FIP200 (Bethyl Labs A301-536A), polyclonal anti-phospho-S6K (Cell Signalling 9250), polyclonal anti-S6K (Cell Signalling 9202), polyclonal anti-phospho-Akt (Cell Signalling 4060),) polyclonal anti-LC3 (Abcam ab48394). Polyclonal anti-ATG13 was generated using the peptide sequence LAVHEKNVREFDAFVETLQ [18]. The secondary antibodies used are Alexa Fluor® 680 Goat Anti-Mouse (Invitrogen, A-21058), HRP-conjugated sheep anti-mouse (GE Healthcare) or HRP-conjugated sheep anti-rabbit (GE Healthcare). "
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