The SZT2 Interactome Unravels New Functions of the KICSTOR Complex

Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.

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Rag GTPases and AMPK/TSC2/Rheb mediate the differential regulation of mTORC1 signaling in response to alcohol and leucine

AJP Cell Physiology ◽

10.1152/ajpcell.00407.2011 ◽

2012 ◽

Vol 302 (10) ◽

pp. C1557-C1565 ◽

Cited By ~ 21

Author(s):

Ly Q. Hong-Brown ◽

C. Randell Brown ◽

Abid A. Kazi ◽

Maithili Navaratnarajah ◽

Charles H. Lang

Keyword(s):

Protein Synthesis ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Inhibitory Effect ◽

Ampk Signaling ◽

Mtorc1 Signaling ◽

Ampk Activity ◽

Coordinate Changes ◽

Rag Gtpase ◽

Signaling Components

Leucine (Leu) and insulin both stimulate muscle protein synthesis, albeit at least in part via separate signaling pathways. While alcohol (EtOH) suppresses insulin-stimulated protein synthesis in cultured myocytes, its ability to disrupt Leu signaling and Rag GTPase activity has not been determined. Likewise, little is known regarding the interaction of EtOH and Leu on the AMPK/TSC2/Rheb pathway. Treatment of myocytes with EtOH (100 mM) decreased protein synthesis, whereas Leu (2 mM) increased synthesis. In combination, EtOH suppressed the anabolic effect of Leu. The effects of EtOH and Leu were associated with coordinate changes in the phosphorylation state of mTOR, raptor, and their downstream targets 4EBP1 and S6K1. As such, EtOH suppressed the ability of Leu to activate these signaling components. The Rag signaling pathway was activated by Leu but suppressed by EtOH, as evidenced by changes in the interaction of Rag proteins with mTOR and raptor. Overexpression of constitutively active (ca)RagA and caRagC increased mTORC1 activity, as determined by increased S6K1 phosphorylation. Furthermore, the caRagA-caRagC heterodimer blocked the inhibitory effect of EtOH. EtOH and Leu produced differential effects on AMPK signaling. EtOH enhanced AMPK activity, resulting in increased TSC2 (S1387) and eEF2 phosphorylation, whereas Leu had the opposite effect. EtOH also decreased the interaction of Rheb with mTOR, and this was prevented by Leu. Collectively, our results indicate that EtOH inhibits the anabolic effects that Leu has on protein synthesis and mTORC1 activity by modulating both Rag GTPase function and AMPK/TSC2/Rheb signaling.

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SMALL GTPASE ARF6 ACTIVATES mTORCl-DEPENDENT SIGNALING PATHWAY AND STIMULATES GLIOBLASTOMA CELL GROWTH

Russian Journal of Biotherapy ◽

10.17650/1726-9784-2015-14-2-91-98 ◽

2015 ◽

Vol 14 (2) ◽

pp. 91-98

Author(s):

A. Yu. Zhuravskaya ◽

A. V. Komelkov ◽

E. M. Tchevkina

Keyword(s):

Signaling Pathway ◽

Small Gtpase ◽

Negative Regulator ◽

Alternative Mechanism ◽

Glioblastoma Cells ◽

Mtorc1 Signaling ◽

Signaling Activation ◽

Mtor Complex 1 ◽

Dependent Signaling Pathway

In this study we first identified role of small GTPase Arf6 in stimulation of proliferation and autonomous growth of human glioblastoma cells. We revealed that Arf6 activates the mTOR complex 1 (mTORCl) and this process is appeared to be an alternative mechanism of mTORC1-signaling activation, independent of PI3K/Akt-signaling pathway. We also showed that Arf6 is a negative regulator of protein kinase ERK1/2 in glioblastoma cells.

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CIB2 regulates mTORC1 signaling and is essential for autophagy and visual function

Nature Communications ◽

10.1038/s41467-021-24056-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Saumil Sethna ◽

Patrick A. Scott ◽

Arnaud P. J. Giese ◽

Todd Duncan ◽

Xiaoying Jian ◽

...

Keyword(s):

Visual Function ◽

Molecular Mechanisms ◽

Neurodegenerative Disorder ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Negative Regulator ◽

Age Related Macular Degeneration ◽

Age Related ◽

Mtorc1 Signaling ◽

Marked Accumulation

AbstractAge-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aβ, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling—a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to ‘nucleotide empty’ or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.

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Gene Expression Profiles at Moxibustioned Site (ST36): A Microarray Analysis

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/890579 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Hai-Yan Yin ◽

Yong Tang ◽

Sheng-Feng Lu ◽

Ling Luo ◽

Jia-Ping Wang ◽

...

Keyword(s):

Gene Expression ◽

Microarray Analysis ◽

Expression Profiles ◽

Alternative Therapy ◽

Gene Expression Profiles ◽

Biological Processes ◽

Physiological Conditions ◽

Pathological Conditions ◽

Molecular Events ◽

Zusanli Acupoint

As a major alternative therapy in Traditional Chinese Medicine, it has been demonstrated that moxibustion could generate a series of molecular events in blood, spleen, and brain, and so forth. However, what would happen at the moxibustioned site remained unclear. To answer this question, we performed a microarray analysis with skin tissue taken from the moxibustioned site also Zusanli acupoint (ST36) where 15-minute moxibustion stimulation was administrated. The results exhibited 145 upregulated and 72 downregulated genes which responded immediately under physiological conditions, and 255 upregulated and 243 downregulated genes under pathological conditions. Interestingly, most of the pathways and biological processes of the differentially expressed genes (DEGs) under pathological conditions get involved in immunity, while those under physiological conditions are involved in metabolism.

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Dengue Virus Activates the AMP Kinase-mTOR Axis To Stimulate a Proviral Lipophagy

Journal of Virology ◽

10.1128/jvi.02020-16 ◽

2017 ◽

Vol 91 (11) ◽

Cited By ~ 47

Author(s):

Tristan X. Jordan ◽

Glenn Randall

Keyword(s):

Dengue Virus ◽

Viral Replication ◽

Lipid Droplets ◽

Adenosine Monophosphate ◽

Denv Infection ◽

Ampk Signaling ◽

Selective Autophagy ◽

Dependent Inactivation ◽

Mtorc1 Signaling ◽

Infected Cells

ABSTRACT Robust dengue virus (DENV) replication requires lipophagy, a selective autophagy that targets lipid droplets. The autophagic mobilization of lipids leads to increased β-oxidation in DENV-infected cells. The mechanism by which DENV induces lipophagy is unknown. Here, we show that infection with DENV activates the metabolic regulator 5′ adenosine-monophosphate activated kinase (AMPK), and that the silencing or pharmacological inhibition of AMPK activity decreases DENV replication and the induction of lipophagy. The activity of the mechanistic target of rapamycin complex 1 (mTORC1) decreases in DENV-infected cells and is inversely correlated with lipophagy induction. Constitutive activation of mTORC1 by depletion of tuberous sclerosis complex 2 (TSC2) inhibits lipophagy induction in DENV-infected cells and decreases viral replication. While AMPK normally stimulates TSC2-dependent inactivation of mTORC1 signaling, mTORC1 inactivation is independent of AMPK activation during DENV infection. Thus, DENV stimulates and requires AMPK signaling as well as AMPK-independent suppression of mTORC1 activity for proviral lipophagy. IMPORTANCE Dengue virus alters host cell lipid metabolism to promote its infection. One mechanism for altered metabolism is the induction of a selective autophagy that targets lipid droplets, termed lipophagy. Lipophagy mobilizes lipid stores, resulting in enhanced β-oxidation and viral replication. We show here that DENV infection activates and requires the central metabolic regulator AMPK for its replication and the induction of lipophagy. This is required for the induction of lipophagy, but not basal autophagy, in DENV-infected cells.

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The transcription factors TFEB and TFE3 link the FLCN-AMPK signaling axis to innate immune response and pathogen resistance

10.1101/463430 ◽

2018 ◽

Author(s):

Leeanna El-Houjeiri ◽

Elite Possik ◽

Tarika Vijayaraghavan ◽

Mathieu Paquette ◽

José A Martina ◽

...

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Mammalian Cells ◽

Pathogen Resistance ◽

Innate Immune ◽

Negative Regulator ◽

Energy Status ◽

Rapid Reduction ◽

Coupling Energy ◽

Mtorc1 Signaling

AbstractTFEB and TFE3 are transcriptional regulators of the innate immune response, but the mechanisms regulating their activation upon pathogen infection are poorly elucidated. UsingC. elegansand mammalian models, we report that the master metabolic modulator 5’-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN) act upstream of TFEB/TFE3 in the innate immune response, independently of the mTORC1 signaling pathway. In nematodes, loss of FLCN or overexpression of AMPK conferred pathogen resistanceviaactivation of TFEB/TFE3-dependent antimicrobial genes, while ablation of total AMPK activity abolished this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induced TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages was observed upon lipopolysaccharide (LPS) treatment accompanied by an acute AMPK activation and TFEB nuclear localization. These results uncover an ancient, highly conserved and pharmacologically actionable mechanism coupling energy status with innate immunity.

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Immune regulators SIB1 and LSD1 antagonistically regulate PhANGs via the GLK transcription factors

10.1101/2020.11.22.393603 ◽

2020 ◽

Author(s):

Mengping Li ◽

Keun Pyo Lee ◽

Tong Liu ◽

Vivek Dogra ◽

Jianli Duan ◽

...

Keyword(s):

Cell Death ◽

Transcription Factors ◽

Critical Level ◽

Negative Regulator ◽

Chloroplast Biogenesis ◽

Sensor Protein ◽

Primed Cell ◽

Light Harvesting Antenna ◽

Signaling Components ◽

Fine Tune

AbstractGOLDEN2-LIKE (GLK) transcription factors drive the expression of photosynthesis-associated nuclear genes (PhANGs), indispensable for chloroplast biogenesis. We previously demonstrated that the salicylic acid (SA)-induced SIGMA FACTOR-BINDING PROTEIN1 (SIB1), a transcription coregulator and positive regulator of SA-primed cell death, interacts with GLKs. The SIB1-GLK interaction raises the level of light-harvesting antenna proteins in the photosystem II, aggravating photoinhibition and singlet oxygen (1O2) burst. 1O2 then contributes to SA-primed cell death via EXECUTER1 (EX1, 1O2 sensor protein)-mediated retrograde signaling upon reaching a critical level. We now reveal that LESION-SIMULATING DISEASE 1 (LSD1), a transcription coregulator and negative regulator of SA-primed cell death, interacts with GLKs to repress their activities. Consistently, the overexpression of LSD1 represses the expression of PhANGs, but the loss of LSD1 increases their expression. The SA-induced SIB1 then counteractively interacts with GLKs, leading to EX1-mediated cell death. Collectively, we provide a working model that mutually exclusive SA-signaling components SIB1 and LSD1 antagonistically regulate GLKs to fine-tune the expression of PhANGs, priming SA-induced cell death, and sustaining 1O2 homeostasis, respectively.

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Coordination of the leucine-sensing Rag GTPase cycle by leucyl-tRNA synthetase in the mTORC1 signaling pathway

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801287115 ◽

2018 ◽

Vol 115 (23) ◽

pp. E5279-E5288 ◽

Cited By ~ 24

Author(s):

Minji Lee ◽

Jong Hyun Kim ◽

Ina Yoon ◽

Chulho Lee ◽

Mohammad Fallahi Sichani ◽

...

Keyword(s):

Protein Synthesis ◽

Trna Synthetase ◽

Gtp Hydrolysis ◽

Mtorc1 Signaling ◽

Amino Acid Sensing ◽

Gtpase Cycle ◽

Rag Gtpase ◽

Cancer Tissues ◽

Mtorc1 Activation ◽

Hydrolysis Of

A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating “ON” switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an “OFF” switch by controlling GTP hydrolysis of RagB in the Rag GTPase–mTORC1 axis. The LRS–RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP–GDP cycle of the RagD–RagB pair, rather than the RagC–RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD–RagB pair can overcome the absence of the RagC–RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD–RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.

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Patched1–ArhGAP36–PKA–Inversin axis determines the ciliary translocation of Smoothened for Sonic Hedgehog pathway activation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1804042116 ◽

2018 ◽

Vol 116 (3) ◽

pp. 874-879 ◽

Cited By ~ 11

Author(s):

Boyan Zhang ◽

Tenghan Zhuang ◽

Qiaoyu Lin ◽

Biying Yang ◽

Xiaowei Xu ◽

...

Keyword(s):

Cell Proliferation ◽

Sonic Hedgehog ◽

Initial Step ◽

Negative Regulator ◽

Individual Development ◽

Sonic Hedgehog Pathway ◽

Shh Pathway ◽

Pathway Activation ◽

Mother Centriole ◽

Signaling Activation

The Sonic Hedgehog (Shh) pathway conducts primarily in the primary cilium and plays important roles in cell proliferation, individual development, and tumorigenesis. Shh ligand binding with its ciliary membrane-localized transmembrane receptor Patched1 results in the removal of Patched1 from and the translocation of the transmembrane oncoprotein Smoothened into the cilium, leading to Shh signaling activation. However, how these processes are coupled remains unknown. Here, we show that the Patched1–ArhGAP36–PKA–Inversin axis determines the ciliary translocation of Smoothened. We find that Patched1 interacts with and stabilizes the PKA negative regulator ArhGAP36 to the centrosome. Activating the Shh pathway results in the removal of ArhGAP36 from the mother centriole and the centrosomal PKA accumulation. This PKA then phosphorylates Inversin and promotes its interaction with and the ciliary translocation of Smoothened. Knockdown of Inversin disrupts the ciliary translocation of Smoothened and Shh pathway activation. These findings reveal a regulatory molecular mechanism for the initial step of Shh pathway activation.

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Amalgam regulates the receptor tyrosine kinase pathway through Sprouty in glial cell development in the Drosophila larval brain

Journal of Cell Science ◽

10.1242/jcs.250837 ◽

2020 ◽

Vol 133 (19) ◽

pp. jcs250837

Author(s):

Majd M. Ariss ◽

Alexander R. Terry ◽

Abul B. M. M. K. Islam ◽

Nissim Hay ◽

Maxim V. Frolov

Keyword(s):

Cell Proliferation ◽

Tyrosine Kinase ◽

Glial Cell ◽

Receptor Tyrosine Kinase ◽

Cell Development ◽

Negative Regulator ◽

Larval Brain ◽

Adhesion Protein ◽

Type Composition ◽

Signaling Activation

ABSTRACTThe receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. Here, we profile the Drosophila larval brain by single-cell RNA-sequencing and identify Amalgam (Ama), which encodes a cell adhesion protein of the immunoglobulin IgLON family, as regulating the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood–brain barrier (BBB), which leads to hemocyte infiltration and neuronal death. We show that Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of the RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPROUTY2 in glioblastoma cell lines, suggesting that the relationship between Ama and Sty is conserved.

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