The identification and analysis of phosphorylation sites on the Atg1 protein kinase

Yeh, Yung‐Hui; Shah, Khyati H.; Chou, Chi-Chi; Hsiao, He-Hsuan; Wrasman, Kristie; Stephan, Joseph S.; Stamatakos, Demetra; Khoo, Kay‐Hooi; Herman, Paul K.

doi:10.4161/auto.7.7.15155

Cited by 25 publications

(36 citation statements)

References 68 publications

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“…16 The kinase domain of Atg1 is located at the N-terminal region (residues 1 to 589), and requires an autophosphorylation event at threonine residue 226 for full activity. 17 The integral membrane protein Atg9 and mammalian BECN1 (the ortholog of yeast Vps30/Atg6) are direct substrates of Atg1 (or the mammalian ortholog ULK1). 18,19 Atg1-mediated phosphorylation events are essential for autophagy and, in the case of Atg9, for the Cvt pathway.…”

Section: Introductionmentioning

confidence: 99%

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Chew¹,

Liu³

et al. 2015

Autophagy

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(2015) Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms, Autophagy, 11:6, 891-905, DOI: 10.1080/15548627.2015 The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1 [CTD]) and Atg13 (Atg13 [CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex. Our data revealed that the intrinsically unstructured C-terminal domain of Atg29 interacts directly with Atg17, whereas Atg17 interacts with Atg13 in 2 distinct intrinsically unstructured regions, including a previously unknown motif that encompasses several putative phosphorylation sites. The Atg1[CTD] crosslinks exclusively to the Atg13[CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13[CTD] and Atg1 [CTD] localize to the tip regions of Atg17-Atg31-Atg29 and do not alter the distinct curvature of this scaffolding subcomplex. This work provides a comprehensive understanding of the subunit interactions in the fully assembled Atg1 core complex, and uncovers the potential role of intrinsically disordered regions in regulating complex integrity.

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Section: Introductionmentioning

confidence: 99%

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Chew¹,

Liu³

et al. 2015

Autophagy

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“…The levels of Thr-226 phosphorylation and total Atg1 protein were determined with a LICOR Biosciences Odyssey infrared imaging system as described under "Experimental Procedures." Atg1-P indicates the position of an autophosphorylated form of Atg1 that migrates anomalously on SDS-polyacrylamide gels (24,28). C, the inhibition of autophagy associated with overexpression of the 550-C fragment of Atg1 was suppressed by the presence of excess Atg13.…”

Section: Discussionmentioning

confidence: 99%

“…Note that different exposure times were used to illustrate the relevant effects of Atg13 and Atg17 overexpression. Atg1-P indicates the position of an autophosphorylated form of Atg1 that migrates anomalously on SDS-polyacrylamide gels (24,28).…”

Section: Atg1 Self-interaction Was Detected Specifically In Cellsmentioning

confidence: 99%

“…This phosphorylation site is conserved in Atg1 orthologs and is likely to be an important site of regulation for all eukaryotes. Finally, recent work has identified a site of phosphorylation within a glycine-rich loop that appears to be inhibitory for kinase activity (28). A major challenge for the field is to develop an understanding of how these different regulatory events are properly coordinated within the eukaryotic cell.…”

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confidence: 99%

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An Atg13 Protein-mediated Self-association of the Atg1 Protein Kinase Is Important for the Induction of Autophagy

Yeh

Shah

Herman

2011

Journal of Biological Chemistry

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Autophagy pathways in eukaryotic cells mediate the turnover of a diverse set of cytoplasmic components, including damaged organelles and abnormal protein aggregates. Autophagy-mediated degradation is highly regulated, and defects in these pathways have been linked to a number of human disorders. The Atg1 protein kinase appears to be a key site of this control and is targeted by multiple signaling pathways to ensure the appropriate autophagic response to changing environmental conditions. Despite the importance of this kinase, relatively little is known about the molecular details of Atg1 activation. In this study we show that Atg13, an evolutionarily conserved regulator of Atg1, promotes the formation of a specific Atg1 self-interaction in the budding yeast, Saccharomyces cerevisiae. The appearance of this Atg1-Atg1 complex is correlated with the induction of autophagy, and conditions that disrupt this complex result in diminished levels of both autophagy and Atg1 kinase activity. Moreover, the addition of a heterologous dimerization domain to Atg1 resulted in elevated kinase activity both in vivo and in vitro. The formation of this complex appears to be an important prerequisite for the subsequent autophosphorylation of Thr-226 in the Atg1 activation loop. Previous work indicates that this modification is necessary and perhaps sufficient for Atg1 kinase activity. Interestingly, this Atg1 self-association does not require Atg17, suggesting that this second conserved regulator might activate Atg1 in a manner mechanistically distinct from that of Atg13. In all, this work suggests a model whereby this self-association stimulates the autophosphorylation of Atg1 within its activation loop.The term autophagy refers to a collection of membrane-trafficking pathways in eukaryotic cells that are responsible for the degradation of cytoplasmic protein and organelles (1-3). During this transport, these cytoplasmic materials are encapsulated within a membrane-bound intermediate and targeted to the lysosome for degradation (4). Defects in these transport pathways have been linked to a variety of human ailments including specific cancers, Crohn disease, and neurological disorders such as Huntington disease (5-8). In many of these conditions autophagy is being considered as a potential point of therapeutic intervention (9 -12). It is, therefore, critical that we develop a thorough understanding of the proteins involved in these transport pathways and the manner in which these degradative activities are regulated.The basic machinery of autophagy is evolutionarily conserved and has been found in all eukaryotic cells (13). The best understood of these degradative pathways is the nonspecific macroautophagy that was initially characterized at the molecular level in the budding yeast, Saccharomyces cerevisiae (2,14,15). This pathway, like most autophagy-based transport, is highly regulated, and the Atg1 protein kinase appears to be a key element of this control (16 -18). This enzyme and its associated partners are targeted by multiple...

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“…Under nutrient rich conditions, the nutrient-sensing target of rapamycin (TOR) kinase and protein kinase A (PKA) inhibit Atg1 complex activation through phosphorylation of Atg1 and Atg13 (Kamada et al, 2000;Kijanska et al, 2010;Kim et al, 2011, Yeh et al, 2011. Starvation induces partial depho-sphorylation of Atg13, and this event activates the Atg1 complex through promoting Atg1 complex formation and Atg1 kinase activity.…”

Section: The Atg1 Kinase Complexmentioning

confidence: 99%

Structural biology of the macroautophagy machinery

Chew

Yip

2014

Front. Biol.

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Macroautophagy is a conserved degradative process mediated through formation of a unique double-membrane structure, the autophagosome. The discovery of autophagy-related (Atg) genes required for autophagosome formation has led to the characterization of approximately 20 genes mediating this process. Recent structural studies of the Atg proteins have provided the molecular basis for their function. Here we summarize the recent progress in elucidating the structural basis for autophagosome formation.

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The identification and analysis of phosphorylation sites on the Atg1 protein kinase

Abstract: (2011) The identification and analysis of phosphorylation sites on the Atg1 protein kinase, Autophagy,7:7,[716][717][718][719][720][721][722][723][724][725][726]

Cited by 25 publications

References 68 publications

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

An Atg13 Protein-mediated Self-association of the Atg1 Protein Kinase Is Important for the Induction of Autophagy

Structural biology of the macroautophagy machinery

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