Phosphorylation Of Tuberous Sclerosis Complex Research Articles

AKT-mediated phosphorylation of TSC2 controls stimulus- and tissue-specific mTORC1 signaling and organ growth.

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates diverse intracellular and extracellular growth signals to regulate cell and tissue growth. How the molecular mechanisms regulating mTORC1 signaling established through biochemical and cell biological studies function under physiological states in specific mammalian tissues are unknown. Here, we characterize a genetic mouse model lacking the 5 phosphorylation sites on the tuberous sclerosis complex 2 (TSC2) protein through which the growth factor-stimulated protein kinase AKT can active mTORC1 signaling in cell culture models. These phospho-mutant mice (TSC2-5A) are developmentally normal but exhibit reduced body weight and the weight of specific organs, such as brain and skeletal muscle, associated with cell intrinsic decreases in growth factor-stimulated mTORC1 signaling. The TSC2-5A mouse model demonstrates that TSC2 phosphorylation is a primary mechanism of mTORC1 activation in some, but not all, tissues and provides a genetic tool to facilitate studies on the physiological regulation of mTORC1.

Abstract 630: The role of AKT pathway in β-catenin mutated hepatocellular carcinoma

Abstract The Wnt/β-catenin and PI3K/AKT pathways are pivotal contributors to the development of the malignant phenotype in cancer. The activated Wnt/β-catenin pathway synergizes with various signaling cascades to propel HCC formation, operating through its downstream effectors. It has been well-established that the mammalian target of rapamycin complex 2 (mTORC2) serves as an upstream regulator of AKT, orchestrating the phosphorylation and activation of AKT. Once activated, AKT phosphorylates and inhibits canonical downstream targets like forkhead box O protein (FOXO) and tuberous sclerosis complex 2 (TSC2). Studies indicated that AKT-mediated phosphorylation of TSC2 activates mTORC1, subsequently suppressing autophagy. However, there are limited data exist on the interplay between Wnt/β-catenin and AKT pathways in HCC. In this study, we sought to explore the role of the AKT pathway in β-catenin activated HCC tumors. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β-catenin (N90-β-catenin) and c-MET in the livers of mice to establish the HCC model (c-Met/β-catenin model). Notably, the knockout of Rictor, a component of the mTORC2 complex, significantly impeded tumor growth, underscoring the pivotal role of AKT in c-Met/β-catenin tumor development. AKT1 and AKT2 are the two main isoforms expressed in the liver. We determined that both AKT1 and AKT2 isoforms are critical for c-Met/β-catenin tumor growth. Intriguingly, simultaneous knockout of Rictor and activation of mTORC1 in the c-Met/β-catenin model resulted in delayed tumor growth, indicating that mTORC1 is not the sole downstream determinant of AKT signaling during c-Met/β-Catenin tumor development. Furthermore, overexpression of the activated FOXO1 (FOXO1AAA) significantly delayed the tumor growth, which highlighted the importance of FOXO1. Given the limited understanding of FOXO1 in HCC, we extended our investigation on FOXO1 to human HCC cell lines. Overexpression of FOXO1AAA notably delayed tumor growth in vitro. RNAseq analysis of FOXO1AAA-overexpressed human HCC cell lines revealed a significant upregulation of "metabolic pathways", suggesting metabolic reprogramming post FOXO1 activation. This study, for the first time, unveils the role of the AKT pathway in β-catenin-mutated HCC growth and highlights the potential therapeutic value of targeting the AKT pathway in patients with β-catenin-mutated HCC. Citation Format: Xue Wang, Xin Chen. The role of AKT pathway in β-catenin mutated hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 630.

Orf virus induces complete autophagy to promote viral replication via inhibition of AKT/mTOR and activation of the ERK1/2/mTOR signalling pathway in OFTu cells

Orf virus (ORFV) is the causative agent of contagious ecthyma, which is an important zoonotic pathogen with a widespread distribution affecting sheep, goats and humans. Our previous research showed that autophagy can be induced in host cells by ORFV infection. However, the exact mechanism of ORFV-induced autophagy remains unknown. In this study, we investigated the underlying mechanisms of autophagy induced by ORFV in OFTu cells and the impact of autophagy on ORFV replication. By using specific autophagy inhibitors and activators, Western blotting, immunofluorescence and transmission electron microscopy imaging, we confirmed that ORFV infection triggered intracellular autophagosome accumulation and the activation of autophagic flux. Moreover, ORFV-induced autophagic activity was found to rely on an increase in the phosphorylation of tuberous sclerosis complex 2 (TSC2) and a decrease in the phosphorylation of mammalian target of rapamycin (mTOR), which is mediated by the suppression of the PI3K/AKT/mTOR signalling pathway and activation of the ERK1/2/mTOR signalling pathway. Furthermore, we investigated the role of mTOR-mediated autophagy during ORFV replication using pharmacological agents and demonstrated that ORFV-induced autophagy correlated positively with viral replication. Taken together, our data reveal the pathways of ORFV-induced autophagy and the impact of autophagy on ORFV replication, providing new insights into ORFV pathogenesis.

AKTs do not translocate to the nucleus upon stimulation but AKT3 can constitutively signal from the nuclear envelope.

AKT is a central signaling protein kinase that plays a role in the regulation of cellular survival metabolism and cell growth, as well as in pathologies such as diabetes and cancer. Human AKT consists of three isoforms (AKT1-3) that may fulfill different functions. Here, we report that distinct subcellular localization of the isoforms directly influences their activity and function. AKT1 is localized primarily in the cytoplasm, AKT2 in the nucleus, and AKT3 in the nucleus or nuclear envelope. None of the isoforms actively translocates into the nucleus upon stimulation. Interestingly, AKT3 at the nuclear envelope is constitutively phosphorylated, enabling a constant phosphorylation of TSC2 at this location. Knockdown of AKT3 induces moderate attenuation of cell proliferation of breast cancer cells. We suggest that in addition to the stimulation-induced activation of the lysosomal/cytoplasmic AKT1-TSC2 pathway, a subpopulation of TSC2 is constitutively inactivated by AKT3 at the nuclear envelope of transformed cells.

PKG oxidation at Cys42 amplifies mTORC1 activation to worsen hypertrophic heart disease

Abstract Rationale Protein kinase G-1a (PKG1a) phosphorylation of tuberous sclerosis complex 2 (TSC2, or tuberin) at serine 1365 (S1365) potently suppresses mTORC1 activation. This results in greater autophagy and limits pathological hypertrophy myocytes and the heart. Cardiac stress also induce oxidation of PKG1a between cysteine 42 residues in homo-monomers, which reduces cardioprotection. Objective We tested the hypothesis that pathological mTORC1 activation is potently amplified by concomitant PKG1a C42-oxidation, and that this is countered by stimulating soluble guanylyl cyclase-1 (GC-1). Methods and results In mice expressing only C42-redox inhibited PKG1a (PKG1aCS), pressure-overload (PO) induces markedly less mTORC1 activation, increasing autophagic flux and reducing protein aggregation, hypertrophy, and dysfunction compared to PO in PKG1aWT mice. Similar results were obtained in cardiomyocytes exposed to endothelin-1. Protection against PO in PKG1aCS mice was similar to PKG1aWT co-treated with the mTORC1-inhibitor everolimus. TSC2 S1365 phosphorylation increased more in PKG1aCS than PKG1aWT myocardium after PO. Knock-in mice with TSC2 S1365A and PKG1aCS mutations, to prevent TSC2 phosphorylation by PKG1a displayed amplified mTORC1, cardiodepression, and mortality after PO as compared to PKG1aCS. Lastly, the marked disparity between PKG1aWT and PKG1aCS PO phenotype for TSC2 S1365 phosphorylation, mTORC1 activation, and cardiac dysfunction is overcome by BAY-602770, a direct stimulator of GC-1. Conclusion Oxidant-induced PKG1a C42 dimerization blunts its attenuation of mTORC1 activity by reducing TSC2 S1365 phosphorylation, thereby contributing to inadequate autophagy and worsened hypertrophic dysfunction. This is ameliorated by direct GC-1 stimulation. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): DFG

PKG1α Cysteine-42 Redox State Controls mTORC1 Activation in Pathological Cardiac Hypertrophy.

Stimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic [mammalian] target of rapamycin complex 1) activation by neurohormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine-42 is also induced by these stressors, which blunts its cardioprotective effects. We tested the dependence of mTORC1 activation on PKG1α C42 oxidation and its capacity to suppress such activation by soluble GC-1 (guanylyl cyclase 1) activation. Cardiomyocytes expressing wild-type (WT) PKG1α (PKG1αWT) or cysteine-42 to serine mutation redox-dead (PKG1αCS/CS) were exposed to ET-1 (endothelin 1). Cells expressing PKG1αWT exhibited substantial mTORC1 activation (p70 S6K [p70 S6 kinase], 4EBP1 [elF4E binding protein-1], and Ulk1 [Unc-51-like kinase 1] phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1αCS/CS expression. Mice with global knock-in of PKG1αCS/CS subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1αWT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1αCS/CS more than PKG1αWT myocardium following PO. TSC2S1365A/S1365A (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1αCS/CS mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1αCS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1αCS/CS after PO and blocked ET-1 stimulated mTORC1 in TSC2S1365A-expressing myocytes. Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.

Tumour kinome re-wiring governs resistance to palbociclib in oestrogen receptor positive breast cancers, highlighting new therapeutic modalities

Combination of CDK4/6 inhibitors and endocrine therapy improves clinical outcome in advanced oestrogen receptor (ER)-positive breast cancer, however relapse is inevitable. Here, we show in model systems that other than loss of RB1 few gene-copy number (CN) alterations are associated with irreversible-resistance to endocrine therapy and subsequent secondary resistance to palbociclib. Resistance to palbociclib occurred as a result of tumour cell re-wiring leading to increased expression of EGFR, MAPK, CDK4, CDK2, CDK7, CCNE1 and CCNE2. Resistance altered the ER genome wide-binding pattern, leading to decreased expression of ‘classical’ oestrogen-regulated genes and was accompanied by reduced sensitivity to fulvestrant and tamoxifen. Persistent CDK4 blockade decreased phosphorylation of tuberous sclerosis complex 2 (TSC2) enhancing EGFR signalling, leading to the re-wiring of ER. Kinome-knockdown confirmed dependency on ERBB-signalling and G2/M–checkpoint proteins such as WEE1, together with the cell cycle master regulator, CDK7. Noteworthy, sensitivity to CDK7 inhibition was associated with loss of ER and RB1 CN. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance and highlights new therapeutic networks that can be exploited upon relapse after CDK4/6 inhibition.

AMPK/TSC2/mTOR pathway regulates replicative senescence of human vascular smooth muscle cells.

Age-associated diseases, including vascular diseases, are on the rise with the increase in the aging population. However, the mechanisms of aging and age-associated vascular dysfunction remain to be fully elucidated. Replicative senescence of vascular smooth muscle cells (VSMCs) contributes to aging as well as age-associated vascular diseases. Rapamycin may delay aging-associated diseases via inhibition of the mammalian target of rapamycin (mTOR), but its role in VSMC aging has remained elusive. The present study investigated the involvement of mTOR signaling in replicative senescence of VSMCs. Replicative senescence was induced in human VSMCs by extended passages and identified by assessing the cell morphology, senescence-associated β-galactosidase activity, and p53 and p21 protein expression. Protein expression and phosphorylation were determined by western blot analysis. Significant senescence of VSMCs was observed in cells subjected to extended passaging (until passage 15). Significant decreases in adenosine monophosphate-activated protein kinase (AMPK)/tuberous sclerosis complex 2 (TSC2) phosphorylation, but significant increases in mTOR/ribosomal protein S6 kinase 1 (S6K1) phosphorylation, were observed in cells with replicative senescence compared with those in young cells. Pre-treatment of VSMCs with AMPK activator and mTOR inhibitor delayed replicative senescence and reversed changes in AMPKα, TSC2, mTOR and S6K1 phosphorylation in senescent VSMCs. The AMPK/TSC2/mTOR/S6K1 signaling axis was found to have an important role in regulating replicative senescence of human VSMCs.

Abstract B72: Signaling and mRNA translation regulation in RSK-deficient LN-18 glioblastoma cell line

Abstract The deregulation of p90 ribosomal S6 kinases (RSKs) seems to be responsible for different oncogenic outcomes in several types of tumors. The effects on mRNA translation regulation via RSKs in glioblastomas (GBMs) were not described so far; thus it is essential to understand the molecular pathways regulated by RSKs that control protein synthesis in GBM. Recent data from our group (unpublished) indicate that different GBM cell lines express distinct levels of the RSK1 isoform, while RSK2 levels are much less variable, and RSK3 and RSK4 are not detected. LN-18 cells show higher levels of RSK1 when compared to A172 or U-118 MG; thus CRISPR/Cas9 system technology was used in this cell line to generate RSK-deficient cells. Briefly, two different sets of small guide RNAs directed to both RSK1 and RSK2 were constructed. LN-18 cells were transfected to express Cas9 along with either one of the constructs targeting RSK1 and/or RSK2. Selection of individual cell clones was followed by Western blotting detection of RSK1 and RSK2 isoforms to identify RSK-deficient cells. We were able to efficiently generate clones with no detection of RSK1 (RSK1-/-), RSK2 (RSK2-/-) with both sets of sgRNAs and cells that had both isoforms knocked out (RSK1-/-/RSK2-/-) only with one of the sgRNA sets. Considering that mTORC1 signaling pathway is the main mediator of RSKs-dependent translation activation, the crosstalk between these pathways and its effects on translation levels were studied in RSKs-deficient cells. Therefore, cells were serum starved for 48 h and stimulated with phorbol-12-myristate-13-acetate (PMA) to activate the Ras-ERK1/2-RSK pathway. Signaling pathways involved in RSK and mTORC1 translation activation were assessed. Interestingly, RSK-deficient cells show lower levels of phosphorylation of Tuberous Sclerosis Complex 2 (TSC-2) at 1798 serine residue, confirming the importance of RSK phosphorylation on this residue and its relevance for evaluation of active RSK levels on cells. In order to describe the influence of RSK1 in the translation initiation rate of GBM cells, polysome profiling was assayed in LN-18, A172, and U-118 MG cells, containing high, low, or nondetected levels of RSK1, respectively. Treatment with PMA for 1, 2, or 4 hours affected translation in GBM cells in a different manner, with the cell lines that display lower levels of RSK1 being less sensitive to the treatment, when compared to LN-18 cells. Polysome profiling was also performed with CRISPR/Cas9-generated cells. RSK1-/- and RSK2-/- cells showed reduced translation initiation levels in basal conditions; however, translation initiation was stimulated after PMA treatment, probably because the remaining isoform is enough to elicit this response. Surprisingly, translation levels were comparable between control cells and RSK1-/-/RSK2-/- cells, suggesting the presence of a compensatory mechanism during the generation of double knockout cells. Of note, this same response could be observed in cells that display alterations in the elongation phase, as it can occur by the lack of phosphorylation of eEF2 kinase in the absence of RSKs. In conclusion, the generation of RSKs-deficient GBM-cell line by the CRISPR/Cas9 system is a useful model for studying and characterizing RSK effects on signaling and translation in GBM cells. Supported by FAPESP. Citation Format: Danielle Nascimento, Martín Roffé, Vilma Regina Martins. Signaling and mRNA translation regulation in RSK-deficient LN-18 glioblastoma cell line [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B72.

BRSK2 induced by nutrient deprivation promotes Akt activity in pancreatic cancer via downregulation of mTOR activity.

Neoplastic cells in pancreatic ductual adenocarcinoma (PDAC) survive in an energy-deprived milieu, and hyper-activation of Akt is thought to contribute to the neoplastic cell survival in PDAC. Kras activating mutations, common in PDAC, was believed to be the major driver of Akt activation. However, the inhibitor to Kras was not therapeutic for PDAC patients. This implied that PDAC cells might harbor an intrinsic merit that strengthens Akt activity. Here we showed that BRSK2, a serine/threonine-protein kinase of AMPK family, was induced by nutrient deprivation in PDAC cells and suppressed mTORC1 activity via phosphorylation of tuberous sclerosis complex 2 (TSC2). The suppression of mTORC1 activity in PDAC results in a dominant loss of feedback inhibition on Akt activity by mTORC1, consequently enhancing cell survival. This finding indicates that the intrinsic molecular merit that BRSK2 provides is a survival advantage to PDAC cells and strengthens the invasiveness of these neoplastic cells in energy-deprived environments.

Cryptotanshinone activates AMPK-TSC2 axis leading to inhibition of mTORC1 signaling in cancer cells

BackgroundCryptotanshinone (CPT), a fat-soluble phenanthraquinone from Salvia miltiorrhiza Bunge, has been demonstrated to inhibit phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a couple of direct downstream effectors of the mammalian target of rapamycin complex 1 (mTORC1), resulting in cancer cell arrested in G0 phase and subsequent inhibition of proliferation. However, its concrete molecular mechanism about how CPT inhibits mTORC1 signaling pathway is unclear.Methodsone solution was used to check cell viability and western blotting for determining expression of the indicated proteins. Molecular docking was performed to assess the binding of CPT with mTOR. The co-immunoprecipitation assay was to analyze whether CPT could disrupt the mTORC1 and TSC1/TSC2 complex. Recombinant adenoviral dominant-negative AMPKα was used to downregulate expression of AMPKα and lentiviral AMPK and TSC2 to silence the AMPK and TSC2 in Rh30 cells.ResultsPrimarily, Rh30 cells expressing rapamycin-resistant mutant mTOR are also sensitive to CPT, while the molecular docking result for CPT binding to mTOR is negative, suggesting that CPT inhibition of mTORC1 is different from rapamycin. Then the related proteins of PTEN-PI3K pathway was proved not to be affected, but the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was activated by a concentration- and time- dependent manner, meaning that it may be associated with AMPK. Further results indicated that compound C, inhibitor of AMPK, could clearly reversed CPT inhibitory effect on Rh30 cells, and dominant-negative AMPK in cancer cells conferred resistance to CPT inhibition of 4E-BP1 and phosphorylation of S6K1, as well as sh-AMPK. Furthermore, compared with AMPK-positive MEF cells, AMPK-negative MEF cells are less sensitive to CPT by the findings that 4E-BP1 and phosphorylation of S6K1 express comparatively more. Additionally, phosphorylation of tuberous sclerosis complex 2 (TSC2) was activated under the treatment of CPT, and down-expression of TSC2 by shRNA slightly recovered expression of 4E-BP1 and phosphorylation of S6K1, while co-immunoprecipitation of TSC2 did not alter expression of TSC1 by CPT.ConclusionCPT inhibiting mTORC1 pathway was mostly due to activation of AMPK-TSC2 axis rather than specific binding to mTORC1. CPT is a potent anticancer agent targeting AMPK.

Hyperglycemia-Induced Oxidative-Nitrosative Stress Induces Inflammation and Neurodegeneration via Augmented Tuberous Sclerosis Complex-2 (TSC-2) Activation in Neuronal Cells.

Diabetes is a systemic disease mainly characterized by chronic hyperglycemia and with extensive and long-lasting spiteful complications in central nervous systems (CNS). Astrocytes play an important role in the defense mechanism of CNS, with great ability of withstanding accumulation of toxic substances. Apart from functional disorders, hyperglycemia leads to slow progressive structural abnormalities in the CNS through oxidative stress pathways. However, the molecular mechanism by which neurons die under oxidative stress induced by high glucose (HG) remains largely unclear. Here, we report that HG-induced inflammation and neurodegeneration in brain tissues, brain astrocytes (C6), and pheochromocytoma (PC-12) cells are cultured in HG conditions. Our results show that the increases in phosphorylation of Akt and ERK1/2MAPK are associated with increased accumulations of reactive oxygen species (ROS) in neuronal cells, which simultaneously enhanced phosphorylations of tuberous sclerosis complex-2 (TSC-2) and mammalian target of rapamycin (mTOR) in the diabetic brain and in HG-exposed neuronal cells. Pharmacologic inhibition of Akt or ERK1/2 or siRNA-mediated gene silencing of TSC-2 suppressed the strong downregulation of TSC-2-mTOR activation. Findings of this study also demonstrate that HG resulted in phosphorylation of NF-κB, coinciding with the increased production of inflammatory mediators and activation of neurodegenerative markers. Pretreatment of cells with antioxidants, phosphoinositide3-kinase (PI3-K)/Akt, and ERK1/2 inhibitors significantly reduced HG-induced TSC-2 phosphorylation and restored NF-κB protein expression leading to decreased production of inflammatory mediators and neurodegenerative markers. These results illustrate that ROS functions as a key signaling component in the regulatory pathway induced by elevated glucose in neuronal cell activation leading to inflammation and neurodegeneration.

Activation of extracellular regulated kinase and mechanistic target of rapamycin pathway in focal cortical dysplasia

Neuropathology of resected brain tissue has revealed an association of focal cortical dysplasia (FCD) with drug-resistant epilepsy (DRE). Recent studies have shown that the mechanistic target of rapamycin (mTOR) pathway is hyperactivated in FCD as evidenced by increased phosphorylation of the ribosomal protein S6 (S6) at serine 240/244 (S(240/244) ), a downstream target of mTOR. Moreover, extracellular regulated kinase (ERK) has been shown to phosphorylate S6 at serine 235/236 (S(235/236) ) and tuberous sclerosis complex 2 (TSC2) at serine 664 (S(664) ) leading to hyperactive mTOR signaling. We evaluated ERK phosphorylation of S6 and TSC2 in two types of FCD (FCD I and FCD II) as a candidate mechanism contributing to mTOR pathway dysregulation. Tissue samples from patients with tuberous sclerosis (TS) served as a positive control. Immunostaining for phospho-S6 (pS6(240/244) and pS6(235/236) ), phospho-ERK (pERK), and phospho-TSC2 (pTSC2) was performed on resected brain tissue with FCD and TS. We found increased pS6(240/244) and pS6(235/236) staining in FCD I, FCD II and TS compared to normal-appearing tissue, while pERK and pTSC2 staining was increased only in FCD IIb and TS tissue. Our results suggest that both the ERK and mTOR pathways are dysregulated in FCD and TS; however, the signaling alterations are different for FCD I as compared to FCD II and TS.

Identification of an AMPK phosphorylation site in Drosophila TSC2 (gigas) that regulate cell growth.

AMP-activated protein kinase (AMPK) is an important metabolic regulator that mediates cellular adaptation to diverse stresses. One of the AMPK substrates, tuberous sclerosis complex 2 (TSC2), was suggested to mediate AMPK-induced silencing of mTOR complex 1 (mTORC1) signaling that is critical for cell growth. However, it is not known whether the AMPK-dependent TSC2 phosphorylation, originally observed in mammalian cells, is conserved in invertebrates. Here we show that energy depletion inhibits mTORC1 signaling through the AMPK-TSC2 axis in Drosophila S2 cells. We have discovered an AMPK phosphorylation site in TSC2-like genes from many different invertebrate species including Drosophila. The site (Ser1338 in Drosophila TSC2) is specifically and efficiently phosphorylated by AMPK in vitro. To evaluate the functional role of this phosphorylation site in vivo, we generated transgenic flies that can express identical amount of either wild-type or phosphorylation-resistant mutant Drosophila TSC2 in a tissue-specific manner. In response to transgenic Sestrin induction, which causes ectopic AMPK activation and subsequent mTORC1 inhibition, wild-type Drosophila TSC2 synergistically reduced tissue growth in the dorsal epithelium of Drosophila wings. However, phosphorylation-resistant mutant Drosophila TSC2 was unable to show such a growth-inhibiting effect, suggesting that this phosphorylation is important for AMPK-dependent regulation of cell growth.

Akt-dependent Activation of mTORC1 Complex Involves Phosphorylation of mTOR (Mammalian Target of Rapamycin) by IκB Kinase α (IKKα)

The serine/threonine protein kinase Akt promotes cell survival, growth, and proliferation through phosphorylation of different downstream substrates. A key effector of Akt is the mammalian target of rapamycin (mTOR). Akt is known to stimulate mTORC1 activity through phosphorylation of tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity. We previously reported that IκB kinase α (IKKα), a component of the kinase complex that leads to NF-κB activation, plays an important role in promoting mTORC1 activity downstream of activated Akt. Here, we demonstrate IKKα-dependent regulation of mTORC1 using multiple PTEN null cancer cell lines and an animal model with deletion of IKKα. Importantly, IKKα is shown to phosphorylate mTOR at serine 1415 in a manner dependent on Akt to promote mTORC1 activity. These results demonstrate that IKKα is an effector of Akt in promoting mTORC1 activity.

Insulin‐dependent and ‐independent regulation of sterol regulatory element‐binding protein‐1c

Insulin‐dependent and ‐independent regulation of sterol regulatory element‐binding protein‐1c

Eccentric contractions increase the phosphorylation of tuberous sclerosis complex‐2 (TSC2) and alter the targeting of TSC2 and the mechanistic target of rapamycin to the lysosome

The goal of this study was to determine whether the mechanical activation of mechanistic target of rapamycin (mTOR) signalling is associated with changes in phosphorylation of tuberous sclerosis complex-2 (TSC2) and targeting of mTOR and TSC2 to the lysosome. As a source of mechanical stimulation, mouse skeletal muscles were subjected to eccentric contractions (ECs). The results demonstrated that ECs induced hyper-phosphorylation of TSC2 and at least part of this increase occurred on residue(s) that fall within RxRxxS/T consensus motif(s). Furthermore, in control muscles, we found that both mTOR and TSC2 are highly enriched at the lysosome. Intriguingly, ECs enhanced the lysosomal association of mTOR and almost completely abolished the lysosomal association of TSC2. Based on these results, we developed a new model that could potentially explain how mechanical stimuli activate mTOR signalling. Furthermore, this is the first study to reveal that the activation of mTOR is associated with the translocation of TSC2 away from the lysosome. Since a large number of signalling pathways rely on TSC2 to control mTOR signalling, our results have potentially revealed a fundamental mechanism via which not only mechanical, but also various other types of stimuli, control mTOR signalling.

Focal adhesion kinase is required for IGF-I-mediated growth of skeletal muscle cells via a TSC2/mTOR/S6K1-associated pathway

Focal adhesion kinase (FAK) is an attachment complex protein associated with the regulation of muscle mass through as-of-yet unclear mechanisms. We tested whether FAK is functionally important for muscle hypertrophy, with the hypothesis that FAK knockdown (FAK-KD) would impede cell growth associated with a trophic stimulus. C₂C₁₂ skeletal muscle cells harboring FAK-targeted (FAK-KD) or scrambled (SCR) shRNA were created using lentiviral transfection techniques. Both FAK-KD and SCR myotubes were incubated for 24 h with IGF-I (10 ng/ml), and additional SCR cells (±IGF-1) were incubated with a FAK kinase inhibitor before assay of cell growth. Muscle protein synthesis (MPS) and putative FAK signaling mechanisms (immunoblotting and coimmunoprecipitation) were assessed. IGF-I-induced increases in myotube width (+41 ± 7% vs. non-IGF-I-treated) and total protein (+44 ± 6%) were, after 24 h, attenuated in FAK-KD cells, whereas MPS was suppressed in FAK-KD vs. SCR after 4 h. These blunted responses were associated with attenuated IGF-I-induced FAK Tyr³⁹⁷ phosphorylation and markedly suppressed phosphorylation of tuberous sclerosis complex 2 (TSC2) and critical downstream mTOR signaling (ribosomal S6 kinase, eIF4F assembly) in FAK shRNA cells (all P < 0.05 vs. IGF-I-treated SCR cells). However, binding of FAK to TSC2 or its phosphatase Shp-2 was not affected by IGF-I or cell phenotype. Finally, FAK-KD-mediated suppression of cell growth was recapitulated by direct inhibition of FAK kinase activity in SCR cells. We conclude that FAK is required for IGF-I-induced muscle hypertrophy, signaling through a TSC2/mTOR/S6K1-dependent pathway via means requiring the kinase activity of FAK but not altered FAK-TSC2 or FAK-Shp-2 binding.

Energy Depletion of Bovine Mammary Epithelial Cells Activates AMPK and Suppresses Protein Synthesis Through Inhibition of mTORC1 Signaling

The molecular mechanisms by which cellular energy status regulates global protein synthesis in mammary epithelial cells have not been characterized. The objective of this study was to examine the effect of AMP-activated protein kinase (AMPK) activation by 2-deoxyglucose on protein synthesis and the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in bovine mammary epithelial cells. Phosphorylation of AMPK at Thr172 increased by 1.4-fold within 5 min, and remained elevated throughout a 30-min time course, in response to 2-deoxyglucose. Global rates of protein synthesis declined by 78% of control values. The decline in protein synthesis was associated with repression of mTORC1 signaling, as indicated by reduced phosphorylation of ribosomal protein S6 kinase 1 and eIF4E binding protein-1 (4E-BP1). Phosphorylation of ER-stress marker eIF2α was also increased but only at 30 min of 2-deoxyglucose exposure. 2-Deoxyglucose increased phosphorylation of tuberous sclerosis complex 2 (TSC2) on AMPK consensus sites but did not change the amount of TSC1 bound to TSC2. Activation of AMPK did not result in changes in the amount of raptor bound to mTOR. The inhibitory effects of AMPK activation on mTORC1 signaling were associated with a marked increase in Ser792 phosphorylation on raptor. Collectively, the results suggest that activation of AMPK represses global protein synthesis in mammary epithelial cells through inhibition of mTORC1 signaling.

Differential Regulation of Protein Synthesis and mTOR Signaling in Skeletal Muscle and Visceral Tissues of Neonatal Pigs After a Meal

Protein synthesis (PS) increases after a meal in neonates, but the time course of the changes in PS in different tissues after a meal is unknown. We aimed to evaluate the changes in tissue PS, mammalian target of rapamycin complex 1 (mTORC1) activation, and proportion of ribosomal protein (rp) mRNAs in polysomes over 4 h after a bolus meal in neonatal pigs (n = 6/group; 5- to 7-d-old). The results show a more sustained increase in PS in glycolytic compared with mixed fiber type muscles and no changes in oxidative muscles. PS increased in liver, jejunum, and pancreas but not in kidney and heart. Feeding did not affect AMP-activated protein kinase or RAS-related GTP binding B activation. Phosphorylation of tuberous sclerosis complex 2, proline-rich Akt substrate of 40 kD, mTOR, eukaryotic initiation factor 4E binding protein, and rp S6 kinase 1 increased in all tissues after feeding. The proportion of mRNAs encoding rp S4 and S8 in liver polysomes increased within 30 min postfeeding. These results suggest that feeding stimulates mTORC1 signaling in muscle and viscera, but mTORC1 activation alone is not sufficient to stimulate PS in all tissues.