Skp2-Mediated RagA Ubiquitination Elicits a Negative Feedback to Prevent Amino-Acid-Dependent mTORC1 Hyperactivation by Recruiting GATOR1

Abstract

Summary The regulation of RagAGTP is important for amino acid-induced mTORC1 activation. Although GATOR1 complex has been identified as a negative regulator for mTORC1 by hydrolyzing RagAGTP, how GATOR1 is recruited to RagA to attenuate mTORC1 signaling remains unclear. Moreover, how mTORC1 signaling is terminated upon amino acid stimulation is also unknown. We show that the recruitment of GATOR1 to RagA is induced by amino acids in an mTORC1-dependent manner. Skp2 E3 ligase drives K63-linked ubiquitination of RagA, which facilitates GATOR1 recruitment and RagAGTP hydrolysis, thereby providing a negative feedback loop to attenuate mTORC1 lysosomal recruitment and prevent mTORC1 hyperactivation. We further demonstrate that Skp2 promotes autophagy, but inhibits cell size and cilia growth through RagA ubiquitination and mTORC1 inhibition. We thereby propose a negative feedback that Skp2-mediated RagA ubiquitination recruits GATOR1 to restrict mTORC1 signaling upon sustained amino acid stimulation, which serves a critical mechanism to maintain proper cellular functions.

Multiple amino acid sensing inputs to mTORC1

“…This observation is inconsistent with a previous study showing that amino acid starvation increases the interaction between GATOR1 and RAGA [50]. SKP2 recruitment to RAGA upon amino acid stimulation is dependent on mTORC1 activity since rapamycin prevents SKP2 recruitment as well as interaction between RAGA and GATOR1 [63]. Jin et al proposed that SKP2-mediated RAGA ubiquitination is a negative feedback mechanism to prevent mTORC1 hyperactivation upon amino acid stimulation.…”

“…However, RNF152 preferentially binds to the already inactive RAGA-GDP, suggesting that RNF152-mediated ubiquitination is not the primary mechanism of RAGA inactivation by GATOR1. Jin et al [63] surprisingly found that amino acids promote SKP2-mediated RAGA interaction with GATOR1. This observation is inconsistent with a previous study showing that amino acid starvation increases the interaction between GATOR1 and RAGA [50].…”

“…Two independent studies demonstrated that RAGA ubiquitination promotes its binding to GATOR1, leading to inactivation of RAGA [63,64]. Deng et al [64] identified RNF152 as the E3 ubiquitin ligase that mediates ubiquitination of RAGA at lysine 142, 220, 230, and 244, while Jin et al [63] found that another E3 ubiquitin ligase, SKP2, mediates ubiquitination of RAGA at lysine 15. Each study showed that the described ubiquitination increases interaction between RAGA and GATOR1, and overexpression of a RAGA ubiquitination-deficient mutant (K142R, K220R, K230R, K244R, or K15R) reduces RAGA-GATOR1 interaction, and thus increases mTORC1 activity.…”

“…Jin et al proposed that SKP2-mediated RAGA ubiquitination is a negative feedback mechanism to prevent mTORC1 hyperactivation upon amino acid stimulation. Both studies showed that MEFs from RNF152-or SKP2-knockout mice have increased mTORC1 activity, presumably due to increased RAGA-GTP levels [63,64]. Curiously, both RNF152 and SKP2 knockout mice are viable [64,65] in contrast to neonatal lethality conferred by RAGA-GTP knockin [66] or SESN knockout [59] (see below).…”

Janus-Faced Sestrin2 Controls ROS and MTOR Signalling Through Two Separate Functional Domains

“…8b,c). Because the Rag GTPases serve as a metabolic switch that can reciprocally regulate AMPK and mTORC1 pathways 51 and the GATOR complex is one of the critical regulators of this switch 52,53 , hSesn2-mediated inhibition of GATOR2 and subsequent activation of GATOR1 (refs 27-29) may be the key signalling event that affects both the AMPK and mTORC1 signalling pathways.…”