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Browsing by Subject "ULK1"

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    Exploring The Interactions And Functions Of The ULK1 Complex In The Autophagy Pathway
    (2014-02) Otto, Neil
    Autophagy, an evolutionarily conserved process through which cellular components or organelles are degraded through lysosomes, is induced when eukaryotic cells are under nutrient starvation or cellular stress conditions. The ULK1 (UNC-51 like kinase 1) complex consisting of ULK1, Atg13, FIP200, and Atg101 plays a key role in mediating cellular nutritional status to the regulation of autophagy. Despite the recent advance in our understanding of the ULK1 functions, how the ULK1 complex regulates autophagy induction remains unclear. Here, we identify that the ULK1 complex interacts with mammalian Atg8 homologs via Atg13 and the interaction is important for autophagosome formation. Through a yeast two-hybrid screen, we identified a clone harboring the full length GATE-16 (Golgi-associated ATPase enhancer of 16 kDa) as an Atg13 binding protein. Through co-immunoprecipitation and in vitro binding assays, we confirmed that Atg13 directly interacts with GATE-16, as well as GABARAP (Gamma-aminobutyric acid receptor-associated protein) and GABARAPL1 (GABA-A receptor-associated protein-like 1), but not LC3B (Microtubule-associated protein1B-light chain 3), via a conserved LC3 interacting region (LIR) near its C-terminus. The Atg13-Atg8 interaction was greatly increased when cells were induced to accumulate protein aggregates or mitochondrial damage, but not by nutrient starvation, implying that the interaction might respond to selective autophagy inducing conditions. The LIR-disrupting mutation of Atg13 suppressed the degradation of p62/sequestosome 1, poly-ubiquitinated protein aggregates, and damaged mitochondria. These results suggest that Atg13 might participate in autophagy, especially selective autophagy, via interacting with GABARAP subfamily Atg8 proteins. p62 is a protein involved in selective autophagy that also interacts with Atg8 proteins via its LIR motif. My study revealed that ULK1 binds and phosphorylates p62. Several phosphorylation sites of p62 were identified by mass spectrometry. Mutational approaches revealed that some of the identified phosphorylations are important for colocalization of p62 with LC3 and for autophagic clearance of mutant huntingtin aggregates. The culmination of this work suggests that the ULK1 complex recruits GABARAP subfamily proteins and phosphorylates p62 in the pathway of autophagy induction.
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    Studies on the regulatory mechanism of the ULK1 complex in the induction of autophagy
    (2012-10) Cao, Jing
    Autophagy, an evolutionarily-conserved cellular process through which organelles and macromolecules are degraded in the lysosome, is induced under nutrient starvation or other unfavorable growth conditions. Unc51-like kinase 1 (ULK1) is a serine/threonine protein kinase that plays a key role in the autophagy induction process, but how ULK1 is regulated by cellular signals for induction of autophagy and how ULK1 regulates the downstream processes in autophagy remain poorly understood. ULK1 interacts with Atg13, focal adhesion kinase family interacting protein of 200 kD (FIP200) and Atg101 to form a large protein complex involved in early steps of the autophagy induction process. To better understand the function of the ULK1 complex, my thesis work has sought to identify binding proteins of the complex. Through a yeast two hybrid screen using a human fetal brain cDNA library with Atg13 as bait, a protein named MCF.2 cell line derived transforming sequence-like 2 (MCF2L2) was identified. Through co-immunoprecipitation and in vitro binding assay, MCF2L2 was determined to directly interact with Atg13 via its N-terminal region independently of ULK1. Knockdown of MCF2L2 inhibited the formation of autophagosome and autophagy flux and led to accumulation of p62/sequestosome-1, a protein degraded through autophagy. Knockdown of MCF2L2 also suppressed the aggregation of WD-repeat protein interacting with phosphoinositides-1, an autophagic isolation membrane marker. MCF2L2 contains a putative Rho-guanine nucleotide exchange factor (GEF) domain in the middle and has a sequence similarity to MCF2L and MCF2, the well-known Rho-GEFs. MCF2L2 overexpression induced a moderate increase in the active forms of Rho GTPases and MCF2L2 colocalized with actin related protein 3, the actin nucleation factor that is regulated by Rho GTPases, implying that MCF2L2 potentially contains GEF activity. MCF2L2 knockdown partially suppressed the distribution of Atg9 from trans-golgi network to the cytoplasm in response to starvation, a process that may depend on actin cytoskeleton. Combined, these results suggest that MCF2L2, as a component of the ULK1 complex, might play an important role in mediating signal transduction between the actin cytoskeleton and autophagy induction.