Abstract
mTOR activation suppresses autophagy by phosphorylating ULK1 at S757 and suppressing its enzymatic activity. Here we report that feedback activation of mTOR in the PI-3 kinase pathway by two p70 S6 kinase (S6K1) inhibitors (PF-4708671 and A77 1726, the active metabolite of an immunosuppressive drug leflunomide) or by S6K1 knockdown did not suppress but rather induced autophagy. Suppression of S6K1 activity led to the phosphorylation and activation of AMPK, which then phosphorylated ULK1 at S555. While mTOR feedback activation led to increased phosphorylation of ULK1 at S757, this modification did not the disrupt ULK1-AMPK interaction nor dampen ULK1 S555 phosphorylation and the induction of autophagy. In addition, inhibition of S6K1 activity led to JNK activation, which also contributed to autophagy. 5Z-7-oxozeaenol, a specific inhibitor of TAK1, or TAK1 siRNA blocked A77 1726-induced activation of AMPK and JNK, and LC3 lipidation. Taken together, our study establishes S6K1 as a key player in the PI-3 kinase pathway to suppress autophagy through inhibiting AMPK and JNK in a TAK1-dependent manner.
Highlights
Macroautophagy is a highly conserved catabolic process characterized by the formation of the double-membraned vesicles, fusion with lysosomes, and degradation of cellular materials
We found that A77 1726 induced AMP-activated protein kinase (AMPK) phosphorylation at T172 and ULK1 phosphorylation at S555 in A375 cells in a dose- (Figure 5A) and time-dependent (Figure 5B) manner. mechanistic target of rapamycin (mTOR) phosphorylates ULK1 at serine 757 (S757) and inhibits its activity as well as autophagy [6, 7]. mTOR is activated by A77 1726 due to the feedback activation of the PI-3 kinase pathway [32]
Our recent study showed that A77 1726 is an inhibitor of p70 S6 kinase (S6K1), and that inhibition of S6K1 activity leads to the feedback activation of the PI-3 kinase pathway [32]
Summary
Macroautophagy (referred as autophagy hereafter) is a highly conserved catabolic process characterized by the formation of the double-membraned vesicles (autophagosomes), fusion with lysosomes, and degradation of cellular materials. Autophagy is activated primarily by nutrient and energy stress. AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) sense energy stress and nutrient depletion, respectively, and play pivotal roles in regulating autophagy [1, 2]. AMPK directly phosphorylates ULK1 at S555 and activates it or indirectly activates ULK1 by inhibiting mTORC1 activity [3,4,5]. MTOR, a serine/threonine kinase www.impactjournals.com/oncotarget that interacts with several adaptor proteins to form the mTOR complex 1 (mTORC1), phosphorylates ULK1/2 at serine 757 (S757), disrupts its interaction with AMPK and prevents it from activating the autophagy pathway [6, 7]. Inactivation of mTORC1 by nutrient insufficiency or by rapamycin, an inhibitor of mTOR, induces autophagy [6, 7].
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