Glycoprotein non-metastatic melanoma protein B promotes tumor growth and is a biomarker for lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare, progressive cystic lung disease affecting almost exclusively female-sexed individuals. The cysts represent regions of lung destruction caused by smooth muscle tumors containing mutations in one of the two tuberous sclerosis (TSC) genes. mTORC1 inhibition slows but does not stop LAM advancement. Furthermore, monitoring disease progression is hindered by insufficient biomarkers. Therefore, new treatment options and biomarkers are needed. LAM cells express melanocytic markers, including glycoprotein non-metastatic melanoma protein B (GPNMB). The function of GPNMB in LAM is currently unknown; however, GPNMB's unique cell surface expression on tumor versus benign cells makes GPNMB a potential therapeutic target, and persistent release of its extracellular ectodomain suggests potential as a serum biomarker. Here, we establish that GPNMB expression is dependent on mTORC1 signaling, and that GPNMB regulates TSC2-null tumor cell invasion in vitro. Further, we demonstrate that GPNMB enhances TSC2-null xenograft tumor growth in vivo, and that ectodomain release is required for this xenograft growth. We also show that GPNMB's ectodomain is released from the cell surface of TSC2-null cells by proteases ADAM10 and 17, and we identify the protease target sequence on GPNMB. Finally, we demonstrate that GPNMB's ectodomain is present at higher levels in LAM patient serum compared to healthy controls and that ectodomain levels decrease with mTORC1 inhibition, making it a potential LAM biomarker.

CBAP regulates the function of Akt-associated TSC protein complexes to modulate mTORC1 signaling.

The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for the phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote the translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.

Non-canonical functions of a mutant TSC2 protein in mitotic division.

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. TSC is caused by mutations in the TSC1 or TSC2 genes, which encode the hamartin/tuberin proteins respectively. These proteins function as a heterodimer that negatively regulates the mechanistic Target of Rapamycin Complex 1 (mTORC1). TSC research has focused on the effects of mTORC1, a critical signaling hub, on regulation of diverse cell processes including metabolism, cell growth, translation, and neurogenesis. However, non-canonical functions of TSC2 are not well studied, and the potential disease-relevant biological mechanisms of mutations affecting these functions are not well understood. We observed aberrant multipolar mitotic division, a novel phenotype, in TSC2 mutant iPSCs. The multipolar phenotype is not meaningfully affected by treatment with the inhibitor rapamycin. We further observed dominant negative activity of the mutant form of TSC2 in producing the multipolar division phenotype. These data expand the knowledge of TSC2 function and pathophysiology which will be highly relevant to future treatments for patients with TSC.

Increased degradation of FMRP contributes to neuronal hyperexcitability in tuberous sclerosis complex.

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder, but new therapies have been impeded by a lack of understanding of the pathological mechanisms. Tuberous sclerosis complex (TSC) and fragile X syndrome are associated with alterations in the mechanistic target of rapamycin (mTOR) and fragile X messenger ribonucleoprotein 1 (FMRP), which have been implicated in the development of ASD. Previously, we observed that transcripts associated with FMRP were down-regulated in TSC2-deficient neurons. In this study, we find that FMRP turnover is dysregulated in TSC2-deficient rodent primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons and is dependent on the E3 ubiquitin ligase anaphase-promoting complex. We also demonstrate that overexpression of FMRP can partially rescue hyperexcitability in TSC2-deficient iPSC-derived neurons. These data indicate that FMRP dysregulation represents an important pathological mechanism in the development of abnormal neuronal activity in TSC and illustrate a molecular convergence between these two neurogenetic disorders.

Glutaredoxin-1 promotes lymphangioleiomyomatosis progression through inhibiting Bim-mediated apoptosis via COX2/PGE2/ERK pathway.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease, characterized by progressive cyst formation and respiratory failure. Clinical treatment with the mTORC1 inhibitor rapamycin could relieve partially the respiratory symptoms, but not curative. It is urgent to illustrate the fundamental mechanisms of TSC2 deficiency to the development of LAM, especially mTORC1-independent mechanisms. Glutaredoxin-1 (Glrx), an essential glutathione (GSH)-dependent thiol-oxidoreductase, maintains redox homeostasis and participates in various processes via controlling protein GSH adducts. Redox signalling through protein GSH adducts in LAM remains largely elusive. Here, we demonstrate the underlying mechanism of Glrx in the pathogenesis of LAM. METHODS: 1. Abnormal Glrx expression in various kinds of human malignancies was identified by the GEPIA tumour database, and the expression of Glrx in LAM-derived cells was detected by real-time quantitative reverse transcription (RT-qPCR) and immunoblot. 2. Stable Glrx knockdown cell line was established to evaluate cellular impact. 3. Cell viability was determined by CCK8 assay. 4. Apoptotic cell number and intracellular reactive oxygen species (ROS) level were quantified by flow cytometry. 5. Cox2 expression and PGE2 production were detected to clarify the mechanism of Bim expression modulated by Glrx. 6. S-glutathionylated p65 was enriched and detected by immunoprecipitation and the direct regulation of Glrx on p65 was determined. 7. The xenograft animal model was established and photon flux was analyzed using IVIS Spectrum. RESULTS: In LAM, TSC2 negatively regulated abnormal Glrx expression and activation in a mTORC1-independent manner. Knockdown of Glrx increased the expression of Bim and the accumulation of ROS, together with elevated S-glutathionylated proteins, contributing to the induction of apoptotic cell death and inhibited cell proliferation. Knockdown of Glrx in TSC2-deficient LAM cells increased GSH adducts on nuclear factor-kappa B p65, which contributed to a decrease in the expression of Cox2 and the biosynthesis of PGE2. Inhibition of PGE2 metabolism attenuated phosphorylation of ERK, which led to the accumulation of Bim, due to the imbalance of its phosphorylation and proteasome degradation. In xenograft tumour models, knockdown of Glrx in TSC2-deficient LAM cells inhibited tumour growth and increased tumour cell apoptosis. CONCLUSIONS: Collectively, we provide a novel redox-dependent mechanism in the pathogenesis of LAM and propose that Glrx may be a beneficial strategy for the treatment of LAM or other TSC-related diseases.

Design of negative-regulating proteins of Rheb/mTORC1 with much-reduced sizes of the tuberous sclerosis protein complex.

The mTORC1 signaling pathway regulates cell growth and metabolism in a variety of organisms from yeast to human, and inhibition of the mTORC1 pathway has the prospect to treat cancer or achieve longevity. The tuberous sclerosis protein complex (TSCC) is a master negative regulator of the mTORC1 signaling pathway through hydrolyzing the GTP loaded on the small GTPase Rheb, which is a key activator of mTOR. However, the large size (~700 kDa) and complex structural organization of TSCC render it vulnerable to degradation and inactivation, thus limiting its potential application. In this work, based on thorough analysis and understanding of the structural mechanism of how the stabilization domain of TSC2 secures the association of TSC2-GAP with Rheb and thus enhances its GAP activity, we designed two proteins, namely SSG-MTM (short stabilization domain and GAP domain-membrane targeting motif) and SSG-TSC1N, which were able to function like TSCC to negatively regulate Rheb and mTORC1, but with much-reduced sizes (~1/15 and ~ 1/9 of the size of TSCC, respectively). Biochemical and cell biological assays demonstrated that these designed proteins indeed could promote the GTPase activity of Rheb to hydrolyze GTP, inhibit the kinase activity of mTORC1, and prevent mTORC1 from down-regulating catabolism and autophagy.

Arginine depletion attenuates renal cystogenesis in tuberous sclerosis complex model.

Cystic kidney disease is a leading cause of morbidity in patients with tuberous sclerosis complex (TSC). We characterize the misregulated metabolic pathways using cell lines, a TSC mouse model, and human kidney sections. Our study reveals a substantial perturbation in the arginine biosynthesis pathway in TSC models with overexpression of argininosuccinate synthetase 1 (ASS1). The rise in ASS1 expression is dependent on the mechanistic target of rapamycin complex 1 (mTORC1) activity. Arginine depletion prevents mTORC1 hyperactivation and cell cycle progression and averts cystogenic signaling overexpression of c-Myc and P65. Accordingly, an arginine-depleted diet substantially reduces the TSC cystic load in mice, indicating the potential therapeutic effects of arginine deprivation for the treatment of TSC-associated kidney disease.

HDAC6-G3BP2 promotes lysosomal-TSC2 and suppresses mTORC1 under ETV4 targeting-induced low-lactate stress in non-small cell lung cancer.

TSC-mTORC1 inhibition-mediated translational reprogramming is a major adaptation mechanism upon many stresses, such as low-oxygen, -ATP, and -amino acids. But how cancer cells hijack the adaptive pathway to survive under low-lactate stress when targeting glycolysis-related signaling remains uncertain. ETV4 is an oncogenic transcription factor frequently dysregulated in human cancer. We previously found that ETV4 is associated with tumor progression and poor prognosis in non-small cell lung cancer (NSCLC). In this study, we report that ETV4 controls HK1 expression and glycolysis-lactate production to activate mTORC1 by relieving TSC2 repression of Rheb in NSCLC cells. Targeting ETV4-induced low-lactate stress is an important input for TSC2 to inhibit mTORC1 and global protein synthesis, while the core stress granule components G3BP2 and HDAC6 are selectively translated. Mechanistically, G3BP2 recruits lysosomal-TSC2 to suppress mTORC1. HDAC6 deacetylates TSC2 to sustain protein stability and associates with G3BP2 to facilitate more recruiting of TSC2 to inactivate mTORC1. In addition, the microtubule retrograde transport activity of HDAC6 drives the aggregate-like perinuclear-mTOR distribution paralleled by lower mTORC1 activity under stress. Thus, HDAC6-G3BP2 is the key complex that promotes lysosomal-TSC2 and suppresses mTORC1 when targeting ETV4, which might represent a critical adaptive mechanism for cell survival under low-lactate challenges.

High glucose-induced increasing reactive nitrogen species accumulation triggered mitochondrial dysfunction, inflammation, and apoptosis in keratinocytes.

Growing evidence indicates that skin injuries are a common complication of diabetes. However, the cellular and molecular mechanisms of high glucose (HG) environment trigger nitrosative stress-mediated inflammation and apoptosis in keratinocytes remains unknown. Here we investigated whether reactive nitrogen species (RNS) induced by HG environment restrain antioxidant activity, and mitochondrial dysfunction leading to inflammation, and apoptosis via stress signaling pathways in keratinocytes. Our results established that the HG environment enhanced the production of nitric oxide (NO) and peroxynitrite anion (ONOO-) by inducible NO synthase (iNOS) in keratinocytes. Overproduction of RNS in HG environment suppress the antioxidants activity leading to mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, decrease in mitochondrial transcription factor A(TFAM), increase in mitochondrial DNA (mtDNA) displacement loop (D-loop) and decrease in glycolytic flux concentration, which was attenuated by pharmacological inhibitors of NO/ONOO-, Nω-Nitro-l-argininemethyl ester hydrochloride (NAME)/hydralazine hydrochloride (Hyd.HCl). Excess production of RNS in HG environment restrained 8-oxoguanine DNA glycosylase-1 (OGG1) expression and increased 8-hydroxydeoxyguanosine (8-OHdG) accumulations in DNA were regulated by NO or ONOO-. Further, HG-induced RNA production caused an increase in the production of inflammatory mediators accompanied by activation of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3 signaling cascade, lipid peroxidation (LPO), and protein carbonylation (PC) reactions followed by breakdown the cell-cell communication and apoptosis. Pre-treatment of cell with NAME/Hyd.HCl, diminished the expression of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3, inflammatory mediators, and attenuated apoptosis in keratinocytes. Together, our results indicated that excess production of RNS in HG environment triggered inflammation and apoptosis, mediated by activation of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3 signaling cascades in keratinocytes.

Lymphangioleiomyomatosis: a metastatic lung disease.

Lymphangioleiomyomatosis (LAM) is a rare disease affecting women, caused by somatic mutations in the TSC1 or TSC2 genes, and driven by estrogen. Similar to many cancers, it is metastatic, primarily to the lung. Despite its monogenetic nature, like many cancers, LAM is a heterogeneous disease. The cellular constituents of LAM are very diverse, including mesenchymal, epithelial, endothelial, and immune cells. LAM is characterized by dysregulation of many cell signaling pathways, distinct populations of LAM cells, and a rich microenvironment, in which the immune system appears to play an important role. This review delineates the heterogeneity of LAM and focuses on the metastatic features of LAM, the deregulated signaling mechanisms and the tumor microenvironment. Understanding the tumor-host interaction in LAM may provide insights into the development of new therapeutic strategies, which could be combinatorial or superlative to Sirolimus, the current U.S. Food and Drug Administration-approved treatment.

Cortical wiring by synapse type-specific control of local protein synthesis.

Neurons use local protein synthesis to support their morphological complexity, which requires independent control across multiple subcellular compartments up to the level of individual synapses. We identify a signaling pathway that regulates the local synthesis of proteins required to form excitatory synapses on parvalbumin-expressing (PV+) interneurons in the mouse cerebral cortex. This process involves regulation of the TSC subunit 2 (Tsc2) by the Erb-B2 receptor tyrosine kinase 4 (ErbB4), which enables local control of messenger RNA {mRNA} translation in a cell type-specific and synapse type-specific manner. Ribosome-associated mRNA profiling reveals a molecular program of synaptic proteins downstream of ErbB4 signaling required to form excitatory inputs on PV+ interneurons. Thus, specific connections use local protein synthesis to control synapse formation in the nervous system.

Ca2+/Calmodulin induces translocation of membrane-associated TSC2 to the nucleus where it suppresses CYP24A1 expression.

Tuberous sclerosis complex 2 (TSC2) is a tumor-suppressor protein. A loss of TSC2 function induces hyperactivation of mechanistic target of rapamycin (mTOR). The C-terminal region of TSC2 contains a calmodulin (CaM) binding region and the CaM-TSC2 interaction contributes to proper mTOR activity. However, other downstream signaling pathways/effectors activated by the CaM-TSC2 complex have not been fully elucidated. In this study, we found that activation of Ca2+/CaM signaling resulted in the translocation of membrane-associated TSC2 to the nucleus and suppressed the transcriptional activity of the vitamin D receptor (VDR). TSC2 was released from the membrane in an activated CaM-dependent state in rat brain and HeLa cells. It subsequently formed a transcriptional complex to partially suppress the transcription of CYP24A1, a well-known VDR target gene. These data suggest, in part, that TSC2 attenuates VDR-associated transcriptional regulation via Ca2+/CaM signaling.

LncRNA PVT-1 promotes osteosarcoma cancer stem-like properties through direct interaction with TRIM28 and TSC2 ubiquitination.

Osteosarcoma, the most common pediatric bone tumor, is an aggressive heterogeneous malignancy defined by complex chromosomal aberrations. Overall survival rates remain at ~70%, but patients with chemoresistant or metastatic disease have extremely poor outcomes of <30%. A subgroup of tumors harbor amplification of chromosome 8q24.2 and increased expression of the oncogenic long noncoding RNA (lncRNA) Plasmacytoma Variant Translocation-1 (PVT-1), which is associated with an extremely poor clinical prognosis. This study demonstrates that PVT-1 is critical for osteosarcoma tumor-initiation potential. Chromatin Hybridization by RNA Purification analysis identified Tripartite-Motif Containing Family 28 (TRIM28) as a novel PVT-1 binding partner. Mechanistically, co-immunoprecipitation studies showed the PVT-1/TRIM28 complex binds and increases SUMOylation of phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34), which leads to enhanced ubiquitination and degradation of tumor suppressor complex 2 (TSC2), thus contributing to increased self-renewal and stem cell phenotypes. Furthermore, we identified that osteosarcoma cells with increased PVT-1 have enhanced sensitivity to the SUMOylation inhibitor, TAK-981. Altogether, this study elucidated a role for PVT-1 in the enhancement of cancer stem-like behaviors, including migration and invasion, in osteosarcoma, and identified the novel PVT-1/TRIM28 axis signaling cascade as a potential therapeutic target for osteosarcoma treatment.

Construction of TSC2 knockout cell line using CRISPR/Cas9 system and demonstration of its effects on NIH-3T3 cells.

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder involving multiple organ systems. TSC2 gene plays an important role in the development of TSC. The most common kidney manifestation of TSC is renal angiomyolipoma (RAML). TSC-RAML is more likely to be bilateral multiple tumors and tends to destroy the renal structure and damages renal function severely. As a result, patients with TSC-RAML often miss the opportunity for surgical treatment when TSC-RAML is diagnosed, causing difficulty in obtaining tumor specimens through surgery. Due to this difficulty, model cell lines must be constructed for scientific research. In this paper, TSC2 was knocked out in NIH-3T3 cell lines by CRISPR/Cas9 system. PCR, WB and mTOR inhibitor drug sensitivity test showed that the TSC2 knockout NIH-3T3 cells were successfully constructed. The ability of proliferation and invasion in TSC2 KO NIH-3T3 cells were higher than those in wild type group. The constructed KO cell line lay the foundation for further study of TSC.

Exosome Release by Glucose Deprivation Is Important for the Viability of TSC-Null Cells.

The control of exosome release is associated with numerous physiological and pathological activities, and that release is often indicative of health, disease, and environmental nutrient stress. Tuberous sclerosis complex (TSC) regulates the cell viability via the negative regulation of the mammalian target of rapamycin complex (mTORC1) during glucose deprivation. However, the mechanism by which viability of TSC-null cells is regulated by mTORC1 inhibition under glucose deprivation remains unclear. Here, we demonstrated that exosome release regulates cell death induced by glucose deprivation in TSC-null cells. The mTORC1 inhibition by rapamycin significantly increased the exosome biogenesis, exosome secretion, and cell viability in TSC-null cells. In addition, the increase in cell viability by mTORC1 inhibition was attenuated by two different types of inhibitors of exosome release under glucose deprivation. Taken together, we suggest that exosome release inhibition might be a novel way for regression of cell growth in TSC-null cells showing lack of cell death by mTORC1 inhibition.

Renal Transcriptome and Metabolome in Mice with Principal Cell-Specific Ablation of the Tsc1 Gene: Derangements in Pathways Associated with Cell Metabolism, Growth and Acid Secretion.

Tuberous sclerosis complex (TSC) is caused by mutations in the hamartin (TSC1) or tuberin (TSC2) genes. Using a mouse model of TSC renal cystogenesis that we have previously described, the current studies delineate the metabolic changes in the kidney and their relation to alterations in renal gene expression. To accomplish this, we compared the metabolome and transcriptome of kidneys from 28-day-old wildtype (Wt) and principal cell-specific Tsc1 KO (Tsc1 KO) mice using targeted 1H nuclear magnetic resonance targeted metabolomic and RNA-seq analyses. The significant changes in the kidney metabolome of Tsc1 KO mice included reductions in the level of several amino acids and significant decreases in creatine, NADH, inosine, UDP-galactose, GTP and myo-inositol levels. These derangements may affect energy production and storage, signal transduction and synthetic pathways. The pertinent derangement in the transcriptome of Tsc1 KO mice was associated with increased collecting duct acid secretion, active cell division and the up-regulation of signaling pathways (e.g., MAPK and AKT/PI3K) that suppress the TSC2 GTPase-activating function. The combined renal metabolome and transcriptome alterations observed in these studies correlate with the unregulated growth and predominance of genotypically normal A-intercalated cells in the epithelium of renal cysts in Tsc1 KO mice.

Raptor knockdown concurrently increases the electrical resistance and paracellular permeability of Caco-2 cell monolayers.

AIMS: As a critical regulatory point of nutrient sensing, growth and metabolism, the mechanistic target of rapamycin complex 1 (mTORC1) is poised to influence intestinal homeostasis under basal conditions and in disease state. Intestinal barrier integrity ensures tissue homeostasis by closely regulating the permeability of the epithelium to lumenal contents. The role of mTORC1 in the regulation of intestinal barrier function and permeability remains to be fully elucidated. MATERIALS AND METHODS: In this study, we employed lentivirus-mediated knockdown of mTORC1 signaling-associated proteins Raptor (regulatory-associated protein of mTOR) and TSC2 (tuberin) to ascertain the effects of constitutive activation or repression of mTORC1 activity on barrier function in Caco-2 cell monolayers. KEY FINDINGS: Results showed that the loss of Raptor concomitantly raised the transepithelial electrical resistance (TEER) and para/transcellular permeability leading to a cell monolayer that is leaky for dextran yet electrically resistant to the movement of ions. Paracellular permeability was linked to the downregulation of tight junction protein expression and enhanced autophagy. Raptor-depleted cells had the highest abundance of myosin binding subunit MYPT1 concomitantly with the lowest abundance of p-MYPT1 (Thr696) and phosphorylated myosin light chain (p-MLC, Ser19) implying that MLC phosphatase activity was increased resulting in MLC relaxation. Although rapamycin suppressed mTORC1 activity and decreased the abundance of tight junction proteins in control cells, rapamycin caused a modest increase of TEER compared to Raptor knockdown. SIGNIFICANCE: The study showed that epithelium paracellular permeability of small molecular weight dextran is dissociated from TEER.

LAM Cells as Potential Drivers of Senescence in Lymphangioleiomyomatosis Microenvironment.

Senescence is a stress-response process characterized by the irreversible inhibition of cell proliferation, associated to the acquisition of a senescence-associated secretory phenotype (SASP), that may drive pathological conditions. Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells, featuring the hyperactivation of the mammalian Target of Rapamycin Complex 1 (mTORC1) for the absence of tuberin expression, cause the disruption of the lung parenchyma. Considering that LAM cells secrete SASP factors and that mTOR is also a driver of senescence, we deepened the contribution of senescence in LAM cell phenotype. We firstly demonstrated that human primary tuberin-deficient LAM cells (LAM/TSC cells) have senescent features depending on mTOR hyperactivation, since their high positivity to SA-ß galactosidase and to phospho-histone H2A.X are reduced by inducing tuberin expression and by inhibiting mTOR with rapamycin. Then, we demonstrated the capability of LAM/TSC cells to induce senescence. Indeed, primary lung fibroblasts (PLFs) grown in LAM/TSC conditioned medium increased the positivity to SA-ß galactosidase and to phospho-histone H2A.X, as well as p21WAF1/CIP1 expression, and enhanced the mRNA expression and the secretion of the SASP component IL-8. Taken together, these data make senescence a novel field of study to understand LAM development and progression.

The non-essential TSC complex component TBC1D7 restricts tissue mTORC1 signaling and brain and neuron growth.

The tuberous sclerosis complex (TSC) 1 and 2 proteins associate with TBC1D7 to form the TSC complex, which is an essential suppressor of mTOR complex 1 (mTORC1), a ubiquitous driver of cell and tissue growth. Loss-of-function mutations in TSC1 or TSC2, but not TBC1D7, give rise to TSC, a pleiotropic disorder with aberrant activation of mTORC1 in various tissues. Here, we characterize mice with genetic deletion of Tbc1d7, which are viable with normal growth and development. Consistent with partial loss of function of the TSC complex, Tbc1d7 knockout (KO) mice display variable increases in tissue mTORC1 signaling with increased muscle fiber size but with strength and motor defects. Their most pronounced phenotype is brain overgrowth due to thickening of the cerebral cortex, with enhanced neuron-intrinsic mTORC1 signaling and growth. Thus, TBC1D7 is required for full TSC complex function in tissues, and the brain is particularly sensitive to its growth-suppressing activities.

Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt.

Tuberous sclerosis complex-2 (TSC2) negatively regulates mammalian target of rapamycin complex 1 (mTORC1), and its activity is reduced by protein kinase B (Akt) and extracellular response kinase (ERK1/2) phosphorylation to activate mTORC1. Serine 1364 (human) on TSC2 bidirectionally modifies mTORC1 activation by pathological growth factors or hemodynamic stress but has no impact on resting activity. We now show this modification biases to ERK1/2 but not Akt-dependent TSC2-mTORC1 activation. Endothelin-1-stimulated mTORC1 requires ERK1/2 activation and is bidirectionally modified by phospho-mimetic (S1364E) or phospho-silenced (S1364A) mutations. However, mTORC1 activation by Akt-dependent stimuli (insulin or PDGF) is unaltered by S1364 modification. Thrombin stimulates both pathways, yet only the ERK1/2 component is modulated by S1364. S1364 also has negligible impact on mTORC1 regulation by energy or nutrient status. In vivo, diet-induced obesity, diabetes, and fatty liver couple to Akt activation and are also unaltered by TSC2 S1364 mutations. This contrasts to prior reports showing a marked impact of both on pathological pressure-stress. Thus, S1364 provides ERK1/2-selective mTORC1 control and a genetic means to modify pathological versus physiological mTOR stimuli.