Kuang Shen

Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1.

Sensing amino acids at the lysosome The mTORC1 protein kinase is a complex of proteins that functions to regulate growth and metabolism. Activity of mTORC1 is sensitive to the abundance of amino acids, but how the sensing of amino acids is coupled to the control of mTORC1 has been unclear. Wang et al. searched for predicted membrane proteins that interacted with regulators of mTORC1. They identified a protein currently known only as SLC38A9. Interaction of SLC38A9 with mTORC1 regulators was sensitive to the presence of amino acids. SLC38A9 has sequence similarity to amino acid transporters. Effects of modulation of SLC38A9 in cultured human cells indicate that it may be the sensor that connects the abundance of arginine and leucine to mTORC1 activity. Science, this issue p. 188 A possible sensor for detecting and signaling amino acid abundance is identified. [Also see Perspective by Abraham] The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase–dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid–sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

A Diverse Array of Cancer-Associated MTOR Mutations Are Hyperactivating and Can Predict Rapamycin Sensitivity

Genes encoding components of the PI3K-AKT-mTOR signaling axis are frequently mutated in cancer, but few mutations have been characterized in MTOR, the gene encoding the mTOR kinase. Using publicly available tumor genome sequencing data, we generated a comprehensive catalog of mTOR pathway mutations in cancer, identifying 33 MTOR mutations that confer pathway hyperactivation. The mutations cluster in six distinct regions in the C-terminal half of mTOR and occur in multiple cancer types, with one cluster particularly prominent in kidney cancer. The activating mutations do not affect mTOR complex assembly, but a subset reduces binding to the mTOR inhibitor DEPTOR. mTOR complex 1 (mTORC1) signaling in cells expressing various activating mutations remains sensitive to pharmacologic mTOR inhibition, but is partially resistant to nutrient deprivation. Finally, cancer cell lines with hyperactivating MTOR mutations display heightened sensitivity to rapamycin both in culture and in vivo xenografts, suggesting that such mutations confer mTOR pathway dependency.

KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1

The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy. Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling. Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease.

The amino acid transporter SLC38A9 is a key component of a lysosomal membrane complex that signals arginine sufficiency to mTORC1

Sensing amino acids at the lysosome The mTORC1 protein kinase is a complex of proteins that functions to regulate growth and metabolism. Activity of mTORC1 is sensitive to the abundance of amino acids, but how the sensing of amino acids is coupled to the control of mTORC1 has been unclear. Wang et al. searched for predicted membrane proteins that interacted with regulators of mTORC1. They identified a protein currently known only as SLC38A9. Interaction of SLC38A9 with mTORC1 regulators was sensitive to the presence of amino acids. SLC38A9 has sequence similarity to amino acid transporters. Effects of modulation of SLC38A9 in cultured human cells indicate that it may be the sensor that connects the abundance of arginine and leucine to mTORC1 activity. Science, this issue p. 188 A possible sensor for detecting and signaling amino acid abundance is identified. [Also see Perspective by Abraham] The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase–dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid–sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

Ragulator and SLC38A9 activate the Rag GTPases through noncanonical GEF mechanisms

Significance Amino acids are basic building blocks for all organisms and are essential for cell growth and proliferation. Cells use a set of protein machines to sense the availability of amino acids to carry out basic responses. In particular, the mTORC1 pathway is a central sensor of amino acids in mammalian cells. Residing on the lysosomal surface upon activation, mTORC1 processes signals from the upstream Rag GTPases and stimulates downstream effectors through phosphorylation cascades. Ragulator and SLC38A9 are key components of the lysosomal branch of the amino acid-sensing machinery upstream of the Rag GTPases, but how they regulate the Rag GTPases at the molecular level remains poorly understood. Here we used kinetic analyses to define their functions. The mechanistic target of rapamycin complex 1 (mTORC1) growth pathway detects nutrients through a variety of sensors and regulators that converge on the Rag GTPases, which form heterodimers consisting of RagA or RagB tightly bound to RagC or RagD and control the subcellular localization of mTORC1. The Rag heterodimer uses a unique “locking” mechanism to stabilize its active (GTPRagA–RagCGDP) or inactive (GDPRagA–RagCGTP) nucleotide states. The Ragulator complex tethers the Rag heterodimer to the lysosomal surface, and the SLC38A9 transmembrane protein is a lysosomal arginine sensor that upon activation stimulates mTORC1 activity through the Rag GTPases. How Ragulator and SLC38A9 control the nucleotide loading state of the Rag GTPases remains incompletely understood. Here we find that Ragulator and SLC38A9 are each unique guanine exchange factors (GEFs) that collectively push the Rag GTPases toward the active state. Ragulator triggers GTP release from RagC, thus resolving the locked inactivated state of the Rag GTPases. Upon arginine binding, SLC38A9 converts RagA from the GDP- to the GTP-loaded state, and therefore activates the Rag GTPase heterodimer. Altogether, Ragulator and SLC38A9 act on the Rag GTPases to activate the mTORC1 pathway in response to nutrient sufficiency.