Nitrogen Source Activates TOR (Target of Rapamycin) Complex 1 via Glutamine and Independently of Gtr/Rag Proteins

The evolutionary conserved TOR complex 1 (TORC1) activates cell growth in response to nutrients. In yeast, TORC1 responds to the nitrogen source via a poorly understood mechanism. Leucine, and perhaps other amino acids, activates TORC1 via the small GTPases Gtr1 and Gtr2, orthologs of the mammalian Rag GTPases. Here we investigate the activation of TORC1 by the nitrogen source and how this might be related to TORC1 activation by Gtr/Rag. The quality of the nitrogen source, as defined by its ability to promote growth and glutamine accumulation, directly correlates with its ability to activate TORC1 as measured by Sch9 phosphorylation. Preferred nitrogen sources stimulate rapid, sustained Sch9 phosphorylation and glutamine accumulation. Inhibition of glutamine synthesis reduces TORC1 activity and growth. Poor nitrogen sources stimulate rapid but transient Sch9 phosphorylation. A Gtr1 deficiency prevents the transient stimulation of TORC1 but does not affect the sustained TORC1 activity in response to good nitrogen sources. These findings suggest that the nitrogen source must be converted to glutamine, the preferred nitrogen source in yeast, to sustain TORC1 activity. Furthermore, sustained TORC1 activity is independent of Gtr/Rag. Thus, the nitrogen source and Gtr/Rag activate TORC1 via different mechanisms.     

Brain-expressed X-linked 2 Is Pivotal for Hyperactive Mechanistic Target of Rapamycin (mTOR)-mediated Tumorigenesis

Frequent alteration of upstream proto-oncogenes and tumor suppressor genes activates mechanistic target of rapamycin (mTOR) and causes cancer. However, the downstream effectors of mTOR remain largely elusive. Here we report that brain-expressed X-linked 2 (BEX2) is a novel downstream effector of mTOR. Elevated BEX2 in Tsc2^−/− mouse embryonic fibroblasts, Pten^−/− mouse embryonic fibroblasts, Tsc2-deficient rat uterine leiomyoma cells, and brains of neuronal specific Tsc1 knock-out mice were abolished by mTOR inhibitor rapamycin. Furthermore, BEX2 was also increased in the liver of a hepatic specific Pten knock-out mouse and the kidneys of Tsc2 heterozygous deletion mice, and a patient with tuberous sclerosis complex (TSC). mTOR up-regulation of BEX2 was mediated in parallel by both STAT3 and NF-κB. BEX2 was involved in mTOR up-regulation of VEGF production and angiogenesis. Depletion of BEX2 blunted the tumorigenesis of cells with activated mTOR. Therefore, enhanced STAT3/NF-κB-BEX2-VEGF signaling pathway contributes to hyperactive mTOR-induced tumorigenesis. BEX2 may be targeted for the treatment of the cancers with aberrantly activated mTOR signaling pathway.     

Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy

Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation.     

TORC2-SGK-1 signaling integrates external signals to regulate autophagic turnover of mitochondria via mtROS

ABSTRACT

Macroautophagy/autophagy is an evolutionarily conserved cellular degradation and recycling process that is tightly regulated by external stimuli, diet, and stress. Our recent findings suggest that in C. elegans, a nutrient sensing pathway mediated by MTORC2 (mechanistic target of rapamycin kinase complex 2) and its downstream effector kinase SGK-1 (serum- and glucocorticoid-inducible kinase homolog 1) suppresses autophagy, involving mitophagy. Induced autophagy/mitophagy in MTORC2-deficient animals slows down development and impairs reproduction independently of the SGK-1 effectors DAF-16/FOXO and SKN-1/NFE2L2/NRF2. In this punctum, we discuss how TORC2-SGK-1 signaling might regulate autophagic turnover and its impact on mitochondrial homeostasis via linking mitochondria-derived reactive oxygen species (mtROS) production to mitophagic turnover.     

Regulation of Mammalian Target of Rapamycin Complex 1 by Bcl-2 and Bcl-XL Proteins

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth and metabolism. Its activity is controlled by various types of signals, including growth factors, nutrients, and stresses. In this study, we show that changes in expression levels of two antiapoptotic proteins, Bcl-2 and Bcl-X_L, also affect mTORC1 signaling activity. In cells overexpressing Bcl-X_L, mTORC1 activity is increased and becomes less sensitive to growth factor or nutrient conditions. In contrast, reduction in expression levels of the two antiapoptotic proteins inhibits mTORC1 signaling activity. Our results suggest that the effect of Bcl-2 and Bcl-X_L on mTORC1 is mediated by FKBP38, an inhibitor of mTORC1. The two proteins compete with mTORC1 for FKBP38 binding and hence alter mTORC1 activity. This study reveals a novel cross-talk between Bcl-2/X_L and mTORC1 signaling, which is likely to contribute to cancer development.     

Functional Characterization of PRKAR1A Mutations Reveals a Unique Molecular Mechanism Causing Acrodysostosis but Multiple Mechanisms Causing Carney Complex

The main target of cAMP is PKA, the main regulatory subunit of which (PRKAR1A) presents mutations in two genetic disorders: acrodysostosis and Carney complex. In addition to the initial recurrent mutation (R368X) of the PRKAR1A gene, several missense and nonsense mutations have been observed recently in acrodysostosis with hormonal resistance. These mutations are located in one of the two cAMP-binding domains of the protein, and their functional characterization is presented here. Expression of each of the PRKAR1A mutants results in a reduction of forskolin-induced PKA activation (measured by a reporter assay) and an impaired ability of cAMP to dissociate PRKAR1A from the catalytic PKA subunits by BRET assay. Modeling studies and sensitivity to cAMP analogs specific for domain A (8-piperidinoadenosine 3′,5′-cyclic monophosphate) or domain B (8-(6-aminohexyl)aminoadenosine-3′,5′-cyclic monophosphate) indicate that the mutations impair cAMP binding locally in the domain containing the mutation. Interestingly, two of these mutations affect amino acids for which alternative amino acid substitutions have been reported to cause the Carney complex phenotype. To decipher the molecular mechanism through which homologous substitutions can produce such strikingly different clinical phenotypes, we studied these mutations using the same approaches. Interestingly, the Carney mutants also demonstrated resistance to cAMP, but they expressed additional functional defects, including accelerated PRKAR1A protein degradation. These data demonstrate that a cAMP binding defect is the common molecular mechanism for resistance of PKA activation in acrodysosotosis and that several distinct mechanisms lead to constitutive PKA activation in Carney complex.     

Rapamycin preserves the follicle pool reserve and prolongs the ovarian lifespan of female rats via modulating mTOR activation and sirtuin expression

To maintain the normal length of female reproductive life, the majority of primordial follicles must be maintained in a quiescent state for later use. In this study, we aimed to study the effects of rapamycin on primordial follicle development and investigate the role of mTOR and sirtuin signaling. Rats were treated every other day with an intraperitoneal injection of rapamycin (5 mg/kg) or vehicle. After 10 weeks of treatment, ovaries were harvested for hematoxylin and eosin (HE) staining, and analysis by immunohistochemistry and Western blotting. HE staining showed that the number and percentage of primordial follicles in the rapamycin-treated group were twice the control group (P < 0.001). Immunohistochemical analysis showed that mTOR and phosphorylated-p70S6K were extensively expressed in surviving follicles with strong staining observed in the cytoplasm of the oocyte. Western blotting showed decreased expression of phosphorylated mTOR and phosphorylated p70S6K in the rapamycin-treated group, and increased the expression of both SIRT1 and SIRT6 compared to the control group (P < 0.05). Taken together, these results suggest that rapamycin may inhibit the transition from primordial to developing follicles and preserve the follicle pool reserve, thus extending the ovarian lifespan of female rats via the modulation of mTOR and sirtuin signalings.     

microRNA-206 is required for osteoarthritis development through its effect on apoptosis and autophagy of articular chondrocytes via modulating the phosphoinositide 3-kinase/protein kinase B-mTOR pathway by targeting insulin-like growth factor-1

microRNA (miR) has been shown to be involved in the treatment of diseases such as osteoarthritis (OA). This study aims to investigate the role of miR-206 in regulating insulin-like growth factor-1 (IGF-1) in chondrocyte autophagy and apoptosis in an OA rat model via the phosphoinositide 3-kinase (P13K)/protein kinase B (AKT)-mechanistic target of rapamycin (mTOR) signaling pathway. Wistar rats were used to establish the OA rat model, followed by the observation of histopathological changes, Mankin score, and the detection of IGF-1-positive expression and tissue apoptosis. The underlying regulatory mechanisms of miR-206 were analyzed in concert with treatment by an miR-206 mimic, an miR-206 inhibitor, or small interfering RNA against IGF-1 in chondrocytes isolated from OA rats. Then, the expression of miR-206, IGF-1, and related factors in the signaling pathway, cell cycle, and apoptosis, as well as inflammatory factors, were determined. Subsequently, chondrocyte proliferation, cell cycle distribution, apoptosis, autophagy, and autolysosome were measured. OA articular cartilage tissue exhibited a higher Mankin score, promoted cell apoptotic rate, increased expression of IGF-1, Beclin1, light chain 3 (LC3), Unc-51-like autophagy activating kinase 1 (ULK1), autophagy-related 5 (Atg5), caspase-3, and Bax, yet exhibited decreased expression of miR-206, P13K, AKT, mTOR, and Bcl-2. Besides, miR-206 downregulated the expression of IGF-1 and activated the P13K/AKT signaling pathway. Moreover, miR-206 overexpression and IGF-1 silencing inhibited the interleukins levels (IL-6, IL-17, and IL-18), cell apoptotic rate, the formation of autolysosome, and cell autophagy while promoting the expression of IL-1β and cell proliferation. The findings from our study provide a basis for the efficient treatment of OA by investigating the inhibitory effects of miR-206 on autophagy and apoptosis of articular cartilage in OA via activating the IGF-1-mediated PI3K/AKT-mTOR signaling pathway.     

The effect of fludrocortisone on the uterine receptivity partially mediated by ERK1/2-mTOR pathway

Implantation of embryos needs endometrial receptivity. Mineralocorticoids is one of the causes influencing the implantation window. This study targeted to evaluation fludrocortisone different properties on endometrial receptivity. The objective of this study was to assess whether treatment with fludrocortisone could impact the expression of diverse genes and proteins that are involved in uterine receptivity in mice. In this study, 40 female adult BALB/c mice were used. The samples were allocated to four groups of ten. Control group (C) received: vehicle; fludrocortisone group (FCA): received 1.5 mg/kg fludrocortisone; PP242 group (PP242): received 30 mg/kg PP242; fludrocortisone+PP242 group (FCA+PP242): received fludrocortisone and PP242. Mice were killed on window implantation day after mating and confirmed pregnancy. The endometrial epithelium of mouse was collected to assess mRNA expression of leukemia inhibitory factor (LIF), mucin-1 (MUC1), heparin-binding epidermal growth factor (HB-EGF), (Msx.1), miRNA Let-7a, and miRNA 223-3p as well as protein expression of extracellular signal-regulated kinase 1/2 (ERK1/2), mammalian target of rapamycin (mTOR), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in the uterine using real-time PCR and western blot, respectively. In comparison with the control group, fludrocortisone administration upregulated the expression of LIF, HB-EGF, Msx.1, miRNA Let-7a, ERK1/2, and mTOR in the epithelial endometrium. The PP242-treated group demonstrated a significant rise in the expression of MUC1, miRNA 223-3p and a remarkable decline in ERK1/2 and p-4E-BP1 levels in comparison with the control group. Combination therapy of (FCA+PP242) resulted in a remarkable rise in LIF, Msx-1, HB-EGF, ERK1/2, and mTOR levels, in comparison with the PP242 group. Furthermore, combination therapy of (FCA+PP242) downregulated the expression of MUC1 in comparison with the PP242-treated group. According to the results, fludrocortisone affected uterine receptivity possibly by means of modulating the expression of genes involved in the uterine receptivity and activation of the ERK1/2-mTOR pathway.     

Structural Conservation of Components in the Amino Acid Sensing Branch of the TOR Pathway in Yeast and Mammals

The highly conserved Rag family GTPases have a role in reporting amino acid availability to the TOR (target of rapamycin) signaling complex, which regulates cell growth and metabolism in response to environmental cues. The yeast Rag proteins Gtr1p and Gtr2p were shown in multiple independent studies to interact with the membrane-associated proteins Gse1p (Ego3p) and Gse2p (Ego1p). However, mammalian orthologs of Gse1p and Gse2p could not be identified. We determined the crystal structure of Gse1p and found it to match the fold of two mammalian proteins, MP1 (mitogen-activated protein kinase scaffold protein 1) and p14, which form a heterodimeric complex that had been assigned a scaffolding function in mitogen-activated protein kinase pathways. The significance of this structural similarity is validated by the recent identification of a physical and functional association between mammalian Rag proteins and MP1/p14. Together, these findings reveal that key components of the TOR signaling pathway are structurally conserved between yeast and mammals, despite divergence of sequence to a degree that thwarts detection through simple homology searches.