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The mTORC1-eIF4F axis controls paused pluripotency

ABSTRACT: Mouse embryonic stem cells (mESCs) can self-renew indefinitely and maintain pluripotency. Inhibition of mechanistic target of rapamycin (mTOR) by the kinase inhibitor INK128 is known to induce paused pluripotency in mESCs cultured with traditional serum/LIF medium (SL), but the underlying mechanisms remain unclear. In this study, we demonstrate that mTOR complex 1 (mTORC1) but not complex 2 (mTORC2) mediates mTOR-inhibition induced paused pluripotency in cells grown in both SL and 2iL medium (GSK3- and MEK-inhibitors and LIF). We also show that mTORC1 regulates self-renewal in both conditions mainly through eIF4F-mediated translation initiation that targets mRNAs of both cytosolic and mitochondrial ribosome subunits. Moreover, inhibition of mitochondrial translation is sufficient to induce paused pluripotency. Interestingly, eIF4F also regulates maintenance of pluripotency in an mTORC1-independent but MEK/ERK-dependent manner in SL, indicating that translation of pluripotency genes is controlled differently in SL and 2iL. Our study reveals a detailed picture of how mTOR governs self-renewal in mESCs and uncovers a context-dependent function of eIF4F in pluripotency regulation.

SUBMITTER: Ms. Xueting Xu

PROVIDER: S-SCDT-EMBOR-2021-53081-T | biostudies-other |

REPOSITORIES: biostudies-other

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The mTORC1-eIF4F axis controls paused pluripotency.

Project description:Mouse embryonic stem cells (mESCs) can self-renew indefinitely and maintain pluripotency. Inhibition of mechanistic target of rapamycin (mTOR) by the kinase inhibitor INK128 is known to induce paused pluripotency in mESCs cultured with traditional serum/LIF medium (SL), but the underlying mechanisms remain unclear. In this study, we demonstrate that mTOR complex 1 (mTORC1) but not complex 2 (mTORC2) mediates mTOR inhibition-induced paused pluripotency in cells grown in both SL and 2iL medium (GSK3 and MEK inhibitors and LIF). We also show that mTORC1 regulates self-renewal in both conditions mainly through eIF4F-mediated translation initiation that targets mRNAs of both cytosolic and mitochondrial ribosome subunits. Moreover, inhibition of mitochondrial translation is sufficient to induce paused pluripotency. Interestingly, eIF4F also regulates maintenance of pluripotency in an mTORC1-independent but MEK/ERK-dependent manner in SL, indicating that translation of pluripotency genes is controlled differently in SL and 2iL. Our study reveals a detailed picture of how mTOR governs self-renewal in mESCs and uncovers a context-dependent function of eIF4F in pluripotency regulation.

| S-EPMC8811634 | biostudies-literature

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly.

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Phosphofructokinases Axis Controls Glucose-Dependent mTORC1 Activation Driven by E2F1.

Project description:Cancer cells rely on mTORC1 activity to coordinate mitogenic signaling with nutrients availability for growth. Based on the metabolic function of E2F1, we hypothesize that glucose catabolism driven by E2F1 could participate on mTORC1 activation. Here, we demonstrate that glucose potentiates E2F1-induced mTORC1 activation by promoting mTORC1 translocation to lysosomes, a process that occurs independently of AMPK activation. We showed that E2F1 regulates glucose metabolism by increasing aerobic glycolysis and identified the PFKFB3 regulatory enzyme as an E2F1-regulated gene important for mTORC1 activation. Furthermore, PFKFB3 and PFK1 were found associated to lysosomes and we demonstrated that modulation of PFKFB3 activity, either by substrate accessibility or expression, regulates the translocation of mTORC1 to lysosomes by direct interaction with Rag B and subsequent mTORC1 activity. Our results support a model whereby a glycolytic metabolon containing phosphofructokinases transiently interacts with the lysosome acting as a sensor platform for glucose catabolism toward mTORC1 activity.

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mTORC1 and CK2 coordinate ternary and eIF4F complex assembly

Project description:mRNA translation plays a major role in homeostasis, whereas its dysregulation underpins a variety of pathological states including cancer, metabolic syndrome and neurological disorders. Ternary complex (TC) and eIF4F complex assembly are two major rate-limiting steps in translation initiation that are thought to be regulated by eIF2α phosphorylation, and the mTOR/4E-BP pathway, respectively2. However, how TC and eIF4F assembly are coordinated remains largely unknown. Using polysome-profiling, we show that on a genome-wide scale mTOR suppresses translation of mRNAs, which are translationally activated under short-term ER stress when TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation, which increases recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β appears to act as a previously unidentified mediator of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation.

2016-04-01 | GSE76766 | GEO

An mTORC1-GRASP55 signaling axis controls unconventional secretion to reshape the extracellular proteome upon stress.

Project description:Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.

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An mTORC1-CK2 axis controls starvation induced ER-phagy via FAM134C

Project description:The degradation of endoplasmic reticulum (ER) via selective autophagy is driven by the ER-phagy receptors that facilitate the incorporation of ER-fragments into nascent autophagosomes. How these receptors are regulated, in response to ER-phagy-inducing stimuluses, is largely unknown. Here we propose that starvation, as well as mTOR inhibition, triggers ER-phagy primarily through the activation of the ER-phagy receptor FAM134C. In physiological, nutrient reach, conditions FAM134C is phosphorylated by the Casein kinase 2 (CK2) protein at specific residues negatively affecting FAM134C interaction with the LC3 proteins, thereby preventing ER-phagy. Pharmacological or starvation-induced mTORC1 inhibition limits phosphorylation of FAM134C by CK2, hence promoting FAM134C activation and ER-phagy. Moreover, inhibition of CK2 or the expression of a phospho-mutant FAM134C protein is sufficient to stimulate ER-phagy. Conversely, starvation induced ER-phagy is inhibited in cells and mice that lack FAM134C or expressing a phospho-mimetic FAM134C protein. Overall, these data describe a new mechanism regulating ER-phagy and provides an example of cargo selectivity mechanism during starvation induced autophagy.

2022-10-14 | PXD030451 | Pride

The FBXW7-SHOC2-Raptor Axis Controls the Cross-Talks between the RAS-ERK and mTORC1 Signaling Pathways.

Project description:FBXW7 is a tumor suppressive E3 ligase, whereas RAS-ERK and mechanistic target of rapamycin kinase (mTORC1) are two major oncogenic pathways. Whether and how FBXW7 regulates these two oncogenic pathways are unknown. Here, we showed that SHOC2, a RAS activator, is a FBXW7 substrate. Growth stimuli trigger SHOC2 phosphorylation on Thr507 by the mitogen-activated protein kinase (MAPK) signal, which facilitates FBXW7 binding for ubiquitylation and degradation. FBXW7-mediated SHOC2 degradation terminates the RAS-MAPK signals and inhibits proliferation. Furthermore, SHOC2 selectively binds to Raptor to competitively inhibit the Raptor-mTOR binding to inactivate mTORC1 and induce autophagy, whereas Raptor binding of SHOC2 inhibits the SHOC2-RAS binding to block the MAPK pathway and proliferation. Finally, SHOC2 is overexpressed in pancreatic cancer, which correlated with poor patient survival. SHOC2 mutations were found in lung cancer tissues with gain-of-function activity. Collectively, the SHOC2-Raptor interaction triggers negative cross-talk between RAS-ERK and mTORC1 pathways, whereas FBXW7 regulates both pathways by targeting SHOC2 for ubiquitylation and degradation.

| S-EPMC6503676 | biostudies-literature

The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity.

Project description:Post-transcriptional modifications can control protein abundance, but the extent to which these alterations contribute to the expression of T helper (TH) lineage-defining factors is unknown. Tight regulation of Bcl6 expression, an essential transcription factor for T follicular helper (TFH) cells, is critical as aberrant TFH cell expansion is associated with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here we show that lack of the SLE risk variant Def6 results in deregulation of Bcl6 protein synthesis in T cells as a result of enhanced activation of the mTORC1-4E-BP-eIF4E axis, secondary to aberrant assembly of a raptor-p62-TRAF6 complex. Proteomic analysis reveals that this pathway selectively controls the abundance of a subset of proteins. Rapamycin or raptor deletion ameliorates the aberrant TFH cell expansion in mice lacking Def6. Thus deregulation of mTORC1-dependent pathways controlling protein synthesis can result in T-cell dysfunction, indicating a mechanism by which mTORC1 can promote autoimmunity.Excessive expansion of the T follicular helper (TFH) cell pool is associated with autoimmune disease and Def6 has been identified as an SLE risk variant. Here the authors show that Def6 limits proliferation of TFH cells in mice via alteration of mTORC1 signaling and inhibition of Bcl6 expression.

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Wnt / Beta-Catenin signalling controls epiblast self-organization in naive and paused states of pluripotency

Project description:ES cells grown in growth factor depleted matrigel could recapitulate the morphogenesis of the epiblast from E4.5 to E5.5. This process is characterized with the establishment of epithelial polarity. We used Next-generation sequencing (NGS) technique to analyze the transcriptional change of ES cells grown in 3D culture conditions in the presence of DMSO, 2i, CH or FGF2/Activin. The comparison of transcriptom between different conditions helps us to understand the molecular mechanism underlying morphogenesis of the epiblast. We found naïve pluripotency factors are kept in a high level in 2i and CH treatment in comparison with DMSO and FGF2/Activin treatment. We identified 5 naïve pluripotency factors which may have potential regulatory function in epithelial polarity. To further analyze these 5 pluripotency factors, we found Esrrb is the core transcriptional factor countering epithelial polarity. We also used Next-generation sequencing (NGS) technique to analyze the transcriptional change of Esrrb delta/delta (+4OHT) and control Esrrb fl/fl (-4OHT) ES cells in the presence of CH. Esrrb delta/delta (+4OHT) cell grown in 3D become polarized at 24 hours, whereas the control Esrrb fl/fl (-4OHT) ES cells are still non-polarized. By analyzing the RNA-Seq data, we found the enrichment of MAPK, Apoptosis, Focal adhesion and Tight junction KEGG pathways in Esrrb delta/delta compared to control Esrrb fl/fl cells. This result suggests the essential function of Esrrb in regulating epithelial polarity.

2020-09-17 | GSE141773 | GEO

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