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AMPK Inhibits ULK1-Dependent Autophagosome Formation and Lysosomal Acidification via Distinct Mechanisms.

Molecular and cellular biology (2018-03-07)
Chinwendu Nwadike, Leon E Williamson, Laura E Gallagher, Jun-Lin Guan, Edmond Y W Chan
ABSTRACT

Autophagy maintains metabolism in response to starvation, but each nutrient is sensed distinctly. Amino acid deficiency suppresses mechanistic target of rapamycin complex 1 (MTORC1), while glucose deficiency promotes AMP-activated protein kinase (AMPK). The MTORC1 and AMPK signaling pathways converge onto the ULK1/2 autophagy initiation complex. Here, we show that amino acid starvation promoted formation of ULK1- and sequestosome 1/p62-positive early autophagosomes. Autophagosome initiation was controlled by MTORC1 sensing glutamine, leucine, and arginine levels together. In contrast, glucose starvation promoted AMPK activity, phosphorylation of ULK1 Ser555, and LC3-II accumulation, but with dynamics consistent with a block in autophagy flux. We studied the flux pathway and found that starvation of amino acid but not of glucose activated lysosomal acidification, which occurred independently of autophagy and ULK1. In addition to lack of activation, glucose starvation inhibited the ability of amino acid starvation to activate both autophagosome formation and the lysosome. Activation of AMPK and phosphorylation of ULK1 were determined to specifically inhibit autophagosome formation. AMPK activation also was sufficient to prevent lysosome acidification. These results indicate concerted but distinct AMPK-dependent mechanisms to suppress early and late phases of autophagy.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Earle′s Balanced Salts, With sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Fetal Bovine Serum, USA origin, Dialyzed by ultrafiltration against 0.15 M NaCl, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Sodium bicarbonate solution, solution (7.5%), sterile-filtered, BioReagent, suitable for cell culture