Abstract
mTOR complex 1 (mTORC1) is a major regulator of cell growth and proliferation that coordinates nutrient inputs with anabolic and catabolic processes. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which directly recruit mTORC1 onto the lysosomal surface, its site of activation. The Rag GTPase heterodimer has a unique architecture that consists of two GTPase subunits, RagA or RagB bound to RagC or RagD. Their nucleotide-loading states are strictly controlled by several lysosomal or cytosolic protein complexes that directly detect and transmit the amino acid signals. GATOR1 (GTPase-activating protein (GAP) activity toward Rags-1), a negative regulator of the cytosolic branch of the nutrient-sensing pathway, comprises three subunits, Depdc5 (DEP domain-containing protein 5), Nprl2 (NPR2-like GATOR1 complex subunit), and Nprl3 (NPR3-like GATOR1 complex subunit), and is a GAP for RagA. GATOR1 binds the Rag GTPases via two modes: an inhibitory mode that holds the Rag GTPase heterodimer and has previously been captured by structural determination, and a GAP mode that stimulates GTP hydrolysis by RagA but remains structurally elusive. Here, using site-directed mutagenesis, GTP hydrolysis assays, coimmunoprecipitation experiments, and structural analysis, we probed the GAP mode and found that a critical residue on Nprl2, Arg-78, is the arginine finger that carries out GATOR1's GAP function. Substitutions of this arginine residue rendered mTORC1 signaling insensitive to amino acid starvation and are found frequently in cancers such as glioblastoma. Our results reveal the biochemical bases of mTORC1 inactivation through the GATOR1 complex.
Highlights
Edited by Alex Toker mTOR complex 1 is a major regulator of cell growth and proliferation that coordinates nutrient inputs with anabolic and catabolic processes
Our results reveal the biochemical bases of mTOR complex 1 (mTORC1) inactivation through the GATOR1 complex
Signals from amino acids, glucose, and growth factors feed into the mTORC1 pathway to regulate and balance anabolic and catabolic processes in cells. mTORC1 senses amino acids through a heterodimeric GTPase unit, the Rag GTPases, which consist of RagA or RagB stably bound to RagC or RagD [3,4,5,6]
Summary
The Rag GTPase heterodimer has a unique architecture that consists of two GTPase subunits, RagA or RagB bound to RagC or RagD Their nucleotide-loading states are strictly controlled by several lysosomal or cytosolic protein complexes that directly detect and transmit the amino acid signals. Using site-directed mutagenesis, GTP hydrolysis assays, coimmunoprecipitation experiments, and structural analysis, we probed the GAP mode and found that a critical residue on Nprl, Arg-78, is the arginine finger that carries out GATOR1’s GAP function Substitutions of this arginine residue rendered mTORC1 signaling insensitive to amino acid starvation and are found frequently in cancers such as glioblastoma. In the resolved GATOR1–Rag structure, Depdc is the only subunit that binds the Rag GTPases in which a -strand on the SHEN domain directly contacts the nucleotide-binding pocket of RagA This contact represents a nonproductive, inhibitory conformation that does not execute the GAP function. Further validation of this catalytic site revealed its evolutionary conservation and importance in switching off mTORC1 signaling upon amino acid starvation
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