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
Source: PubMed


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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Available from: Andrea Longatti, Apr 03, 2014
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    • "There are currently 40 autophagy-related (ATG) genes known in yeast, many of which have mammalian orthologues , and the conserved core Atg proteins fall into several groups. Upon amino acid withdrawal, the mammalian target of rapamycin complex 1 (mTORC1) is inactivated, which removes repression on the ULK (uncoordinated 51-like kinase) complex, which consists of ULK1/2, ATG13, FIP200 and ATG101(Hara et al, 2008; Chan et al, 2009; Hosokawa et al, 2009; Mercer et al, 2009). The ULK1 complex then goes on to activate the autophagy-specific phosphatidylinositol 3 kinase (PtdIns(3)K) complex, which includes ATG14, Beclin1, VPS34 and p150 and nucleates pools of phosphatidylinositol-3- phosphate (PtdIns(3)P) at specific sites called omegasomes on the endoplasmic reticulum (ER) marked by double FYVE domaincontaining protein 1 (DFCP1), where the ER is thought to act as a cradle for autophagosome formation (Axe et al, 2008; Hayashi- Nishino et al, 2009; Yla-Anttila et al, 2009). "
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    ABSTRACT: Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain-containing protein, TBC1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB11-positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi-subunit tethering complex and GEF for RAB1, as an interactor of TBC1D14. TBC1D14 binds to the TRAPP complex via an N-terminal 103 amino acid region, and overexpression of this region inhibits both autophagy and secretory traffic. TRAPPC8, the mammalian orthologue of a yeast autophagy-specific TRAPP subunit, forms part of a mammalian TRAPPIII-like complex and both this complex and TBC1D14 are needed for RAB1 activation. TRAPPC8 modulates autophagy and secretory trafficking and is required for TBC1D14 to bind TRAPPIII. Importantly, TBC1D14 and TRAPPIII regulate ATG9 trafficking independently of ULK1. We propose a model whereby TBC1D14 and TRAPPIII regulate a constitutive trafficking step from peripheral recycling endosomes to the early Golgi, maintaining the cycling pool of ATG9 required for initiation of autophagy.
    Preview · Article · Dec 2015 · The EMBO Journal
    • "The reasons that ULK1 is not essential for murine survival could be (i) ULK2, which shows N50% homology with ULK1 and shows functional redundancy and induce autophagy and/or (ii) existence of ULK1 independent mechanism of autophagy. Furthermore, Chan and co-workers have shown that in HEK293 cells ULK1 was critical for inducing autophagy in response to amino acid starvation (Chan et al., 2009). Therefore, the focus of this study was to analyze ULK1 which is a major regulator of autophagy. "
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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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    ABSTRACT: Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction which controls the major metabolic pathways. Among these is the pathway of autophagy which takes care of the degradation of long-lived proteins and of the elimination of damaged or functionally redundant organelles. Proper functioning of this process is essential for cell survival. Dysregulation of autophagy has been implicated in the etiology of several pathologies. The history of the studies on the interrelationship between amino acids, MTOR signaling and autophagy is the subject of this review. The mechanisms responsible for the stimulation of MTOR-mediated signaling, and the inhibition of autophagy, by amino acids have been studied intensively in the past but are still not completely clarified. Recent developments in this field are discussed.
    Full-text · Article · Jun 2014 · Amino Acids
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