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Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy.

The Journal of biological chemistry (2016-01-24)
Cindy Puente, Ronald C Hendrickson, Xuejun Jiang
ABSTRACT

Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-LC3B antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Phosphatase Inhibitor Cocktail 3, DMSO solution
Sigma-Aldrich
Anti-Atg1/ULK1 antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
Sigma-Aldrich
Anti-ATG13 antibody produced in rabbit, ~1.0 mg/mL, affinity isolated antibody
Millipore
S-protein Agarose
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
MBP, Dephosphorylated
Sigma-Aldrich
Anti-β-Actin antibody, Mouse monoclonal, clone AC-15, purified from hybridoma cell culture