AMP-activated protein kinase (AMPK) is an important metabolic regulator that mediates cellular adaptation to diverse stresses. One of the AMPK substrates, tuberous sclerosis complex 2 (TSC2), was suggested to mediate AMPK-induced silencing of mTOR complex 1 (mTORC1) signaling that is critical for cell growth. However, it is not known whether the AMPK-dependent TSC2 phosphorylation, originally observed in mammalian cells, is conserved in invertebrates. Here we show that energy depletion inhibits mTORC1 signaling through the AMPK-TSC2 axis in Drosophila S2 cells. We have discovered an AMPK phosphorylation site in TSC2-like genes from many different invertebrate species including Drosophila. The site (Ser1338 in Drosophila TSC2) is specifically and efficiently phosphorylated by AMPK in vitro
. To evaluate the functional role of this phosphorylation site in vivo
, we generated transgenic flies that can express identical amount of either wild-type or phosphorylation-resistant mutant Drosophila TSC2 in a tissue-specific manner. In response to transgenic Sestrin induction, which causes ectopic AMPK activation and subsequent mTORC1 inhibition, wild-type Drosophila TSC2 synergistically reduced tissue growth in the dorsal epithelium of Drosophila wings. However, phosphorylation-resistant mutant Drosophila TSC2 was unable to show such a growth-inhibiting effect, suggesting that this phosphorylation is important for AMPK-dependent regulation of cell growth.
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