The Ubiquitination Of RagA GTPase By RNF152 Negatively Regulates MTORC1 Activation

Amount evidence suggest that the TOR signaling pathway is capable of simultaneously sensing different signals,and controlling cell metabolism.In cells,mTORC1 plays a specialized role in regulating the process of various physiological and biochemical,incluiding autophagy,mRNA translation,glycolysis,lipid synthesis and nucleotide synthesisPrevious evidence suggested that mTORC1 can senses diverse signals primarily via two kinds of Ras-related small G proteins,the Rag and Rheb GTPases,both of which act directly upstream of mTORC 1.It was well documented that the activation of RagA is tightly regulated by the GEF,GDI and GAP proteins.The lysosomal Ragulator complex functions as a GEF for RagA in response to amino acid stimulation;Sestrins,negatively regulate the amino acid-sensing pathway by functioning as a GDI protein for RagA.Some studies have demonstrated that GATOR1 negatively regulates amino-acid-induced mTORC1 activation.However,the mechanism by which GATOR1 interacts with and regulates the Rag GTPase complex via amino acid-sensing pathways remains unknown.Ubiquitination is an essential posttranslational modification,which controls almost every process in cell.Ubiquitination is brought about by a three-step enzymatic reaction including ubiquitin-activating enzymes E1,E2,E3.Up to date,accumulating evidence shows that ubiquitination plays an essential role in various signaling pathways,such as the NF-kB,Wnt and Hippo pathways.However,the role of ubiquitination in mTORC1 signaling pathway remain largely unknown,and our results shown that ubiquitination was involved in this pathways.Based on this,our current study mainly focused on the underlying mechanism by which ubiquitination regulates mTORC1 activation.In this study,we found that RagA is modified by polyubiquitination in an amino acid-sensitive manner.And we identified the lysosome-anchored E3 ubiquitin ligase RNF152 as an essential negative regulator of the mTORCl pathway by targeting RagA for K63-linked ubiquitination.Mechanically,we found that RNF152 interacts with and ubiquitinates RagA in an amino acid-sensitive manner.Using the mass spectrum we identified the ubiquitinited sites of RagA mediated by RNF152:lysine 142,220,230,and 244.Our further study found that the ubiquitination by RNF152 generates an anchor on RagA to recruit its inhibitor GATOR1,a GAP complex for Rag GTPases,thus inhibits the activation of RagA and mTORCl.RNF 152 knockout results in the hyperactivation of mTORC1 and protects cells from amino acid starvation-induced autophagy.Taken together,we demonstrate that the lysosome-localized E3 ubiquitin ligase RNF 152 negatively regulates mTORC1 function by targeting RagA GTPase for K-63 polyubiquitination,which promotes the recruitment of its inhibitor GATOR1,a GAP complex for Rag GTPases.The regulation of RNF152-mediated RagA ubiquitination is controlled by amino acids.Thus,our study reveals a previously unknown mechanism for regulation of mTROC1 signaling by RNF 152-mediated K63-linked polyubiquitination of RagA.In this work,we discover a new mechanism of how cells sensing nutritions,and define the ubiquitination as a molecular switch that modulate the activation of mTORCl.mTORC1 has emerged as a critical effector in various physiological or pathological conditions,such as cancer,aging and metabolic diseases.It will be interesting to investigate whether RNF 152 acts as a regulator of mTORC1 in various diseases,such as obesity,diabetes,and other liver-related diseases.