Chapter 11 Monitoring Mammalian Target of Rapamycin (mTOR) Activity
59
Citation
40
Reference
10
Related Paper
Citation Trend
Keywords:
mTORC2
RPTOR
The mechanistic target of rapamycin (mTOR) signals through the mTOR complex 1 (mTORC1) and the mTOR complex 2 to maintain cellular and organismal homeostasis. Failure to finely tune mTOR activity results in metabolic dysregulation and disease. While there is substantial understanding of the molecular events leading mTORC1 activation at the lysosome, remarkably little is known about what terminates mTORC1 signaling. Here, we show that the AAA + ATPase Thorase directly binds mTOR, thereby orchestrating the disassembly and inactivation of mTORC1. Thorase disrupts the association of mTOR to Raptor at the mitochondria-lysosome interface and this action is sensitive to amino acids. Lack of Thorase causes accumulation of mTOR-Raptor complexes and altered mTORC1 disassembly/re-assembly dynamics upon changes in amino acid availability. The resulting excessive mTORC1 can be counteracted with rapamycin in vitro and in vivo. Collectively, we reveal Thorase as a key component of the mTOR pathway that disassembles and thus inhibits mTORC1.
mTORC2
RPTOR
Cite
Citations (4)
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr kinase that comprises two complexes, termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 phosphorylates S6K1 at Thr 389, whereas mTORC2 phosphorylates AKT at Ser 473 to promote cell growth. As the mTOR name implies it is the target of natural product called rapamycin, a clinically approved drug used to treat human disease. Short-term rapamycin treatment inhibits the kinase activity of mTORC1 but not mTORC2. However, the ATP-competitive catalytic mTOR inhibitor Torin1 was identified to inhibit the kinase activity of both mTORC1 and mTORC2. Here, we report that H89 (N-(2-(4-bromocinnamylamino) ethyl)-5-isoquinolinesulfonamide), a well-characterized ATP-mimetic kinase inhibitor, renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors across multiple cell lines. Moreover, H89 prevented the dephosphorylation of AKT and S6K1 under nutrient depleted conditions. PKA and other known H89-targeted kinases do not alter the phosphorylation status of S6K1 and AKT. Pharmacological inhibition of some phosphatases also enhanced S6K1 and AKT phosphorylation. These findings suggest a new target for H89 by which it sustains the phosphorylation status of S6K1 and AKT, resulting in mTOR signaling.
mTORC2
RPTOR
Cite
Citations (8)
mTORC2
RPTOR
Cite
Citations (59)
The target of rapamycin (TOR) is a highly conserved serine/threonine kinase that forms two structurally and functionally distinct complexes, TORC1 and TORC2. In response to nutrients, growth factors and cellular energy, mammalian TOR (mTOR) controls cell growth and metabolism. mTOR plays an important role in metabolic organs, particularly in the liver, to mediate whole-body energy homeostasis. Dysregulation of mTOR signaling is associated with the development of several metabolic diseases such as diabetes, obesity, and cancer. Here, we review the role of hepatic mTORC1 and mTORC2 in linking metabolism, cancer development, and circadian rhythm-related processes.
mTORC2
RPTOR
Nutrient Sensing
Sirolimus
Cite
Citations (2)
AbstractThe mammalian target of rapamycin (mTOR) is centrally involved in growth, survival and metabolism. In cancer, mTOR is frequently hyperactivated and is a clinically validated target for drug development. Until recently, we have relied largely on the use of rapamycin to study mTOR function and its anticancer potential. Recent insights now indicate that rapamycin is a partial inhibitor of mTOR through allosteric inhibition of mTOR complex-1 (mTORC1) but not mTOR complex-2 (mTORC2). Both the mechanism of action and the cellular response to mTORC1 inhibition by rapamycin and related drugs may limit the effectiveness of these compounds as antitumor agents. We and others have recently reported the discovery of second-generation ATP-competitive mTOR kinase inhibitors (TKIs) that bind to the active sites of mTORC1 and mTORC2, thereby targeting mTOR signaling function globally (see refs. 1-4). The discovery of specific, active-site mTOR inhibitors has opened a new chapter in the 40-plus year old odyssey that began with the discovery of rapamycin from a soil sample collected on Easter Island (see Vézina C, et al. J Antibiot 1975). Here, we discuss recent studies that highlight the emergence of rapamycin-resistant mTOR function in protein synthesis, cell growth, survival and metabolism. It is shown that these rapamycin-resistant mTOR functions are profoundly inhibited by TKIs. A more complete suppression of mTOR global signaling network by the new inhibitors is expected to yield a deeper and broader antitumor response in the clinic.
mTORC2
RPTOR
Sirolimus
Cite
Citations (157)
The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation in response to growth factor and nutrient signaling. Consequently, this kinase is implicated in metabolic diseases including cancer and diabetes, so there is great interest in understanding the complete spectrum of mTOR-regulated networks. mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, and whereas the natural product rapamycin inhibits only a subset of mTORC1 functions, recently developed ATP-competitive mTOR inhibitors have revealed new roles for both complexes. A number of studies have highlighted mTORC1 as a regulator of lipid homeostasis. We show that the ATP-competitive inhibitor PP242, but not rapamycin, significantly down-regulates cholesterol biosynthesis genes in a 4E-BP1–dependent manner in NIH 3T3 cells, whereas S6 kinase 1 is the dominant regulator in hepatocellular carcinoma cells. To identify other rapamycin-resistant transcriptional outputs of mTOR, we compared the expression profiles of NIH 3T3 cells treated with rapamycin versus PP242. PP242 caused 1,666 genes to be differentially expressed whereas rapamycin affected only 88 genes. Our analysis provides a genomewide view of the transcriptional outputs of mTOR signaling that are insensitive to rapamycin.
mTORC2
RPTOR
Cite
Citations (106)
The mammalian target of rapamycin (mTOR) plays a central role in the regulation of cell growth. mTOR exists in two distinct complexes, mTORC1 and mTORC2. mTORC1 is known to receive inputs from multiple signaling pathways, including growth factors, nutrients, and cellular energy levels to stimulate protein synthesis by phosphorylating key translation regulators such as ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E binding protein 1 (4EBP1). In contrast, mTORC2 regulates cell morphology and also phosphorylates AKT. Recent studies have established that dysregulated mTOR activity is associated with several hamartoma syndromes and metabolic disorders. The mechanism of mTOR regulation by numerous intracellular signaling pathways and the function of mTOR in physiological regulation will be discussed.
mTORC2
RPTOR
Ribosomal protein s6
Cite
Citations (0)
mTORC2
RPTOR
RHEB
Nutrient Sensing
Sirolimus
Cite
Citations (6)
<div>Abstract<p>The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic component of two evolutionarily conserved signaling complexes. mTOR signaling complex 1 (mTORC1) is a key regulator of growth factor and nutrient signaling. S6 kinase is the best-characterized downstream effector of mTORC1. mTOR signaling complex 2 (mTORC2) has a role in regulating the actin cytoskeleton and activating Akt through S473 phosphorylation. Herein, we show that mTOR is phosphorylated differentially when associated with mTORC1 and mTORC2 and that intact complexes are required for these mTORC-specific mTOR phosphorylations. Specifically, we find that mTORC1 contains mTOR phosphorylated predominantly on S2448, whereas mTORC2 contains mTOR phosphorylated predominantly on S2481. Using S2481 phosphorylation as a marker for mTORC2 sensitivity to rapamycin, we find that mTORC2 formation is in fact rapamycin sensitive in several cancer cell lines in which it had been previously reported that mTORC2 assembly and function were rapamycin insensitive. Thus, phospho-S2481 on mTOR serves as a biomarker for intact mTORC2 and its sensitivity to rapamycin. [Cancer Res 2009;69(5):1821–7]</p></div>
mTORC2
RPTOR
Cite
Citations (0)
mTOR is a major biological switch, coordinating an adequate response to changes in energy uptake (amino acids, glucose), growth signals (hormones, growth factors) and environmental stress. mTOR kinase is highly conserved through evolution from yeast to man and in both cases, controls autophagy and cellular translation in response to nutrient stress. mTOR kinase is the catalytic component of two distinct multiprotein complexes called mTORC1 and mTORC2. In addition to mTOR, mTORC1 contains Raptor, mLST8 and PRAS40. mTORC2 contains mTOR, Rictor, mSIN1 and Protor-1. mTORC1 activates p70S6K, which in turn phosphorylates the ribosomal protein S6 and 4E-BP1, both involved in protein translation. mTORC2 activates AKT directly by phosphorylating Serine 473. pAKT(S473) phosphorylates TSC2 (tuberin) and inactivates it, preventing its association with TSC1 (hamartin) and the inhibition of Rheb, an activator of mTOR. pAKT also phosphorylates PRAS40, releasing it from the mTORC1 complex, increasing its kinase activity. Finally, AKT regulates FOXO3 phosphorylation, sequestering it in the cytosol in an inactive state.
mTORC2
RHEB
RPTOR
Cite
Citations (81)