SUMMARY mTORC1 promotes cell growth in response to nutrients and growth factors. Insulin activates mTORC1 through the PI3K-Akt pathway, which inhibits the TSC1-TSC2-TBC1D7 complex (the TSC complex) to turn on Rheb, an essential activator of mTORC1. However, the mechanistic basis of how this pathway integrates with nutrient-sensing pathways is unknown. We demonstrate that insulin stimulates acute dissociation of the TSC complex from the lysosomal surface, where subpopulations of Rheb and mTORC1 reside. The TSC complex associates with the lysosome in a Rheb-dependent manner, and its dissociation in response to insulin requires Akt-mediated TSC2 phosphorylation. Loss of the PTEN tumor suppressor results in constitutive activation of mTORC1 through the Akt-dependent dissociation of the TSC complex from the lysosome. These findings provide a unifying mechanism by which independent pathways affecting the spatial recruitment of mTORC1 and the TSC complex to Rheb at the lysosomal surface serve to integrate diverse growth signals.

The class I phosphoinositide 3-kinase (PI3K) - mechanistic target of rapamycin complex 1 (mTORC1) signaling network directs cellular metabolism and growth. Activation of mTORC1, which is composed of mTOR, Raptor, mLST8, PRAS40, and DEPTOR, depends on the Rag and Rheb GTPases, and requires signals from amino acids, glucose, oxygen, energy (ATP), and growth factors (including cytokines and hormones such as insulin). Here we discuss the signal transduction mechanisms through which growth factor-responsive PI3K signaling activates mTORC1. We focus on how PI3K-dependent activation of Akt and spatial regulation of the TSC complex (composed of TSC1, TSC2, and TBC1D7) switches on Rheb at the lysosome, where mTORC1 is activated. Integration of PI3K- and amino acid-dependent signals upstream of mTORC1 at the lysosome is detailed in a working model. A coherent understanding of the PI3K-mTORC1 network is imperative as its dysregulation has been implicated in diverse pathologies including cancer, diabetes, autism, and aging.

Flux through metabolic pathways is inherently sensitive to the levels of specific substrates and products, but cellular metabolismis also managed by integrated control mechanisms that sense the nutrient and energy status of a cell or organism. The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), a protein kinase complex ubiquitous to eukaryotic cells, has emerged as a critical signaling node that links nutrient sensing to the coordinated regulation of cellular metabolism. Here, the role of mTORC1 as a conduit between cellular growth conditions and the anabolic processes that promote cell growth is discussed. The emerging network of signaling pathways by which mTORC1 integrates systemic signals, in the form of secreted growth factors, with local signals, in the form of cellular nutrients (amino acids, glucose, and oxygen) and energy (ATP) is detailed. Our expanding understanding of the exquisite regulatory network upstream of mTORC1 provides molecular insights into the integrated sensing mechanisms by which diverse cellular signals converge to control cell physiology.

SUMMARY The tuberous sclerosis complex (TSC) tumor suppressors form the TSC1-TSC2 complex, which limits cell growth in response to poor growth conditions. Through its GTPase-activating protein (GAP) activity toward Rheb, this complex inhibits the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), a key promoter of cell growth. Here, we identify and biochemically characterize TBC1D7 as a stably-associated and ubiquitous third core subunit of the TSC1-TSC2 complex. We demonstrate that the TSC1-TSC2-TBC1D7 (TSC-TBC) complex is the functional complex that senses specific cellular growth conditions and possesses Rheb-GAP activity. Sequencing analyses of samples from TSC patients suggest that TBC1D7 is unlikely to represent TSC3. TBC1D7 knockdown decreases the association of TSC1 and TSC2 leading to decreased Rheb-GAP activity, without effects on the localization of TSC2 to the lysosome. Like the other TSC-TBC components, TBC1D7 knockdown results in increased mTORC1 signaling, delayed induction of autophagy, and enhanced cell growth under poor growth conditions.

The mammalian target of rapamycin (mTOR) is a protein kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. Both complexes phosphorylate a hydrophobic motif on downstream protein kinases, which contributes to the activation of these kinases. mTOR complex 1 (mTORC1) phosphorylates S6K1, while mTORC2 phosphorylates Akt. The TSC1-TSC2 complex is a critical negative regulator of mTORC1. However, how mTORC2 is regulated and whether the TSC1-TSC2 complex is involved are unknown. We find that mTORC2 isolated from a variety of cells lacking a functional TSC1-TSC2 complex is impaired in its kinase activity toward Akt. Importantly, the defect in mTORC2 activity in these cells can be separated from effects on mTORC1 signaling and known feedback mechanisms affecting insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Our data also suggest that the TSC1-TSC2 complex positively regulates mTORC2 in a manner independent of its GTPase-activating protein activity toward Rheb. Finally, we find that the TSC1-TSC2 complex can physically associate with mTORC2 but not mTORC1. These data demonstrate that the TSC1-TSC2 complex inhibits mTORC1 and activates mTORC2, which through different mechanisms promotes Akt activation.

The mammalian target of rapamycin (mTOR) functions within two distinct complexes (mTORC1 and mTORC2) to control cell growth, proliferation, survival, and metabolism. While there has been great progress in our understanding of mTORC1 regulation, the signaling mechanisms that regulate mTORC2 have not been defined. In this study, we use liquid chromatography-tandem mass spectrometry analyses to identify 21 phosphorylation sites on the core mTORC2 component Rictor. We find that one site, T1135, undergoes growth factor-responsive phosphorylation that is acutely sensitive to rapamycin and is phosphorylated downstream of mTORC1. We find that Rictor-T1135 is directly phosphorylated by the mTORC1-dependent kinase S6K1. Although this phosphorylation event does not affect mTORC2 integrity or in vitro kinase activity, expression of a phosphorylation site mutant of Rictor (T1135A) in either wild-type or Rictor null cells causes an increase in the mTORC2-dependent phosphorylation of Akt on S473. However, Rictor-T1135 phosphorylation does not appear to regulate mTORC2-mediated effects on SGK1 or PKC␣. While the precise molecular mechanism affecting Akt is unknown, phosphorylation of T1135 stimulates binding of Rictor to 14-3-3 proteins. We provide evidence that Rictor-T1135 phosphorylation acts in parallel with other mTORC1-dependent feedback mechanisms, such as those affecting IRS-1 signaling to PI3K, to regulate the response of Akt to insulin.

During fetal prostate development, Sonic hedgehog (Shh) expression by the urogenital sinus epithelium activates Gli-1 expression in the adjacent mesenchyme and promotes outgrowth of the nascent ducts. Shh signaling is down-regulated at the conclusion of prostate ductal development. However, a survey of adult human prostate tissues reveals substantial levels of Shh signaling in normal, hyperplasic, and malignant prostate tissue. In cancer specimens, the Shh expression is localized to the tumor epithelium, whereas Gli-1 expression is localized to the tumor stroma. Tight correlation between the levels of Shh and Gli-1 expression suggests active signaling between the tissue layers. To determine whether Shh-Gli-1 signaling could be functionally important for tumor growth and progression, we performed experiments with the LNCaP xenograft tumor model and demonstrated that: 1). Shh expressed by LNCaP tumor cells activates Gli-1 expression in the tumor stroma, 2). genetically engineered Shh overexpression in LNCaP cells leads to increased tumor stromal Gli-1 expression, and 3). Shh overexpression dramatically accelerates tumor growth. These data suggest that hedgehog signaling from prostate cancer cells to the stroma can elicit the expression of paracrine signals, which promote tumor growth.

Although it is known that mTOR complex 2 (mTORC2) functions upstream of Akt, the role of this protein kinase complex in cancer is not well understood. Through an integrated analysis of cell lines, in vivo models and clinical samples, we demonstrate that mTORC2 is frequently activated in glioblastoma (GBM), the most common malignant primary brain tumor of adults. We show that the common activating epidermal growth factor receptor (EGFR) mutation (EGFRvIII) stimulates mTORC2 kinase activity, which is partially suppressed by PTEN. mTORC2 signaling promotes GBM growth and survival, and activates NF-κB. Importantly, this mTORC2-NF-κB pathway renders GBM cells and tumors resistant to chemotherapy in a manner independent of Akt. These results highlight the critical role of mTORC2 in GBM pathogenesis, including through activation of NF-κB downstream of mutant EGFR, leading to a previously unrecognized function in cancer chemotherapy resistance. These findings suggest that therapeutic strategies targeting mTORC2, alone or in combination with chemotherapy, will be effective in cancer.