mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6

Abstract

Autophagy is important in the basal or stress-induced clearance of bulk cytosol, damaged organelles, pathogens and selected proteins by specific vesicles, the autophagosomes. Following mTOR (mammalian target of rapamycin) inhibition, autophagosome formation is primed by the ULK1 and the beclin-1-Vps34-AMBRA1 complexes, which are linked together by a scaffold platform, the exocyst. Although several regulative steps have been described along this pathway, few targets of mTOR are known, and the cross-talk between ULK1 and beclin 1 complexes is still not fully understood. We show that under non-autophagic conditions, mTOR inhibits AMBRA1 by phosphorylation, whereas on autophagy induction, AMBRA1 is dephosphorylated. In this condition, AMBRA1, interacting with the E3-ligase TRAF6, supports ULK1 ubiquitylation by LYS-63-linked chains, and its subsequent stabilization, self-association and function. As ULK1 has been shown to activate AMBRA1 by phosphorylation, the proposed pathway may act as a positive regulation loop, which may be targeted in human disorders linked to impaired autophagy.

Chaperone-like protein p32 regulates ULK1 stability and autophagy

“…ULK1 is a relatively stable protein and is subject to proteasome-mediated degradation. Post-translational modifications including K63-linked ubiquitylation 16,17 and phosphorylation [18][19][20] have been reported to modulate the rates of ULK1 turnover and kinase activity in different cellular contexts. Hsp90 and Cdc37 have been shown to regulate ULK1 stability and activity by forming complex with ULK1, which subsequently influences Atg13-mediated mitophagy.…”

“…ULK1 has been suggested to undergo proteasome-mediated degradation. 17,21 We, therefore, tested whether ULK1 reduction in p32-depleted cells could be attributed to increased proteolysis of ULK1. Indeed, proteasomal inhibitor MG132 treatment completely blocked downregulation of ULK1 in p32-ablated cells (Figure 2e), demonstrating that depletion of p32 leads to increased proteasomal degradation of ULK1.…”

“…Previous studies reported that ULK1 is a K63-linked but not K48-linked ubiquitylated protein. 16,17 We investigated whether p32 regulates ULK1 stability via modulating its ubiquitylation. Unlike previous studies, when we used ubiquitin mutants Ub-K48 and Ub-K63, which contain arginine substitutions on all their lysine residues except the one at position 48 and 63, respectively, we found ULK1 is subject to both K48-linked and K63-linked ubiquitylation in vivo (Figure 3a).…”

“…p32 regulates ULK1 stability and autophagy H Jiao et al TRAF6 is the only known E3 ligase that mediates ULK1 K63 ubiquitylation and its subsequent stabilization. 17 We, therefore, tested whether there is an interplay between TRAF6 and p32 in regulating the steady state of ULK1. As both TRAF6 and p32 could bind to ULK1, we wondered whether these two molecules may form protein complex.…”

Autophagy regulation: RNF2 targets AMBRA1

“…Altogether, these findings suggest that the role of WASH in autophagy is dependent on its subcellular localization and its partners in intracellular membranes. some formation via its E3 ligase activity with DDB1-CUL4A to enhance Beclin 1 association with VPS34 [7] and with TRAF6 to stabilize ULK1, which acts in a complex upstream of the PI3K complex 1 in autophagy [9]. Finally, the study by Xia et al emphasizes that autophagy must be tightly regulated to avoid deleterious effects on cell homeostasis.…”