ABSTRACT
Fumarate is an oncometabolite. However, the mechanism underlying fumarate-exerted tumorigenesis remains unclear. Here, utilizing human type2 papillary renal cell carcinoma (PRCC2) as a model, we show that fumarate accumulates in cells deficient in fumarate hydratase (FH) and inhibits PTEN to activate PI3K/AKT signaling. Mechanistically, fumarate directly reacts with PTEN at cysteine 211 (C211) to form S-(2-succino)-cysteine. Succinated C211 occludes tethering of PTEN with the cellular membrane, thereby diminishing its inhibitory effect on the PI3K/AKT pathway. Functionally, re-expressing wild-type FH or PTEN C211S phenocopies an AKT inhibitor in suppressing tumor growth and sensitizing PRCC2 to sunitinib. Analysis of clinical specimens indicates that PTEN C211 succination levels are positively correlated with AKT activation in PRCC2. Collectively, these findings elucidate a non-metabolic, oncogenic role of fumarate in PRCC2 via direct post-translational modification of PTEN and further reveal potential stratification strategies for patients with FH loss by combinatorial AKTi and sunitinib therapy.
Subject(s)
Carcinoma, Papillary , Carcinoma, Renal Cell , Fumarates , Kidney Neoplasms , PTEN Phosphohydrolase , Carcinogenesis , Carcinoma, Papillary/drug therapy , Carcinoma, Papillary/enzymology , Carcinoma, Papillary/genetics , Carcinoma, Papillary/metabolism , Carcinoma, Renal Cell/drug therapy , Carcinoma, Renal Cell/enzymology , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/metabolism , Cysteine/metabolism , Drug Resistance, Neoplasm , Fumarate Hydratase/genetics , Fumarate Hydratase/metabolism , Fumarates/pharmacology , Humans , Kidney Neoplasms/drug therapy , Kidney Neoplasms/enzymology , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , PTEN Phosphohydrolase/antagonists & inhibitors , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics , Sunitinib/pharmacologyABSTRACT
Despite a long appreciation for the role of nonsense-mediated mRNA decay (NMD) in destroying faulty, disease-causing mRNAs and maintaining normal, physiologic mRNA abundance, additional effectors that regulate NMD activity in mammalian cells continue to be identified. Here, we describe a haploid-cell genetic screen for NMD effectors that has unexpectedly identified 13 proteins constituting the AKT signaling pathway. We show that AKT supersedes UPF2 in exon-junction complexes (EJCs) that are devoid of RNPS1 but contain CASC3, defining an unanticipated insulin-stimulated EJC. Without altering UPF1 RNA binding or ATPase activity, AKT-mediated phosphorylation of the UPF1 CH domain at T151 augments UPF1 helicase activity, which is critical for NMD and also decreases the dependence of helicase activity on ATP. We demonstrate that upregulation of AKT signaling contributes to the hyperactivation of NMD that typifies Fragile X syndrome, as exemplified using FMR1-KO neural stem cells derived from induced pluripotent stem cells.
Subject(s)
Nonsense Mediated mRNA Decay , Proto-Oncogene Proteins c-akt , Animals , Codon, Nonsense/genetics , Exons/genetics , Mammals/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA Helicases/genetics , RNA Helicases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/metabolismABSTRACT
Recent work suggests that cholesterol metabolism impacts innate immune responses against infection. However, the key enzymes or the natural products and mechanisms involved are not well elucidated. Here, we have shown that upon DNA and RNA viral infection, macrophages reduced 7-dehydrocholesterol reductase (DHCR7) expression. DHCR7 deficiency or treatment with the natural product 7-dehydrocholesterol (7-DHC) could specifically promote phosphorylation of IRF3 (not TBK1) and enhance type I interferon (IFN-I) production in macrophages. We further elucidated that viral infection or 7-DHC treatment enhanced AKT3 expression and activation. AKT3 directly bound and phosphorylated IRF3 at Ser385, together with TBK1-induced phosphorylation of IRF3 Ser386, to achieve IRF3 dimerization. Deletion of DHCR7 and the DHCR7 inhibitors including AY9944 and the chemotherapy drug tamoxifen promoted clearance of Zika virus and multiple viruses in vitro or in vivo. Taken together, we propose that the DHCR7 inhibitors and 7-DHC are potential therapeutics against emerging or highly pathogenic viruses.
Subject(s)
Dehydrocholesterols/metabolism , Interferon Regulatory Factor-3/metabolism , Interferon Type I/biosynthesis , Macrophages/immunology , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Vesicular Stomatitis/immunology , A549 Cells , Animals , Cell Line , Cholesterol/metabolism , Enzyme Activation/immunology , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Oxidoreductases Acting on CH-CH Group Donors/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , RAW 264.7 Cells , RNA Interference , RNA, Small Interfering/genetics , Vesicular stomatitis Indiana virus/immunologyABSTRACT
AKT is a serine/threonine kinase that plays an important role in metabolism, cell growth, and cytoskeletal dynamics. AKT is activated by two kinases, PDK1 and mTORC2. Although the regulation of PDK1 is well understood, the mechanism that controls mTORC2 is unknown. Here, by investigating insulin receptor signaling in human cells and biochemical reconstitution, we found that insulin induces the activation of mTORC2 toward AKT by assembling a supercomplex with KRAS4B and RHOA GTPases, termed KARATE (KRAS4B-RHOA-mTORC2 Ensemble). Insulin-induced KARATE assembly is controlled via phosphorylation of GTP-bound KRAS4B at S181 and GDP-bound RHOA at S188 by protein kinase A. By developing a KARATE inhibitor, we demonstrate that KRAS4B-RHOA interaction drives KARATE formation. In adipocytes, KARATE controls insulin-dependent translocation of the glucose transporter GLUT4 to the plasma membrane for glucose uptake. Thus, our work reveals a fundamental mechanism that activates mTORC2 toward AKT in insulin-regulated glucose homeostasis.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Glucose/metabolism , Insulin/metabolism , Mechanistic Target of Rapamycin Complex 2/chemistry , rhoA GTP-Binding Protein/chemistry , 3T3-L1 Cells , Adipocytes/cytology , Animals , Cell Membrane/metabolism , Cytoskeleton/metabolism , Dictyostelium , Glucose Transporter Type 4/metabolism , Guanosine Diphosphate/chemistry , Guanosine Triphosphate/chemistry , HEK293 Cells , Humans , Mice , Phosphorylation , Protein Transport , Proto-Oncogene Proteins c-akt/chemistry , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/metabolismABSTRACT
Group 3 innate lymphoid cells (ILC3s) sense environmental signals that are critical for gut homeostasis and host defense. However, the metabolite-sensing G-protein-coupled receptors that regulate colonic ILC3s remain poorly understood. We found that colonic ILC3s expressed Ffar2, a microbial metabolite-sensing receptor, and that Ffar2 agonism promoted ILC3 expansion and function. Deficiency of Ffar2 in ILC3s decreased their in situ proliferation and ILC3-derived interleukin-22 (IL-22) production. This led to impaired gut epithelial function characterized by altered mucus-associated proteins and antimicrobial peptides and increased susceptibility to colonic injury and bacterial infection. Ffar2 increased IL-22+ CCR6+ ILC3s and influenced ILC3 abundance in colonic lymphoid tissues. Ffar2 agonism differentially activated AKT or ERK signaling and increased ILC3-derived IL-22 via an AKT and STAT3 axis. Our findings suggest that Ffar2 regulates colonic ILC3 proliferation and function, and they identify an ILC3-receptor signaling pathway modulating gut homeostasis and pathogen defense.
Subject(s)
Immunity, Innate , Immunity, Mucosal , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Lymphocytes/immunology , Lymphocytes/metabolism , Receptors, Cell Surface/metabolism , Animals , Biomarkers , Cytokines/metabolism , Disease Susceptibility , Gastrointestinal Microbiome/immunology , Gene Expression , Humans , Immunomodulation , Intestinal Mucosa/pathology , Lymphocyte Activation/immunology , Mice , Mice, Knockout , Proto-Oncogene Proteins c-akt , Receptors, Cell Surface/agonists , STAT3 Transcription Factor/metabolismABSTRACT
Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.
Subject(s)
Mammary Neoplasms, Animal/enzymology , Mammary Neoplasms, Animal/pathology , Proto-Oncogene Proteins c-akt/metabolism , Animals , Carcinogenesis/pathology , Female , Gene Deletion , Humans , Insulin/metabolism , Isoenzymes/metabolism , Neoplasm Metastasis , Neutrophils/metabolism , Receptor, ErbB-2/metabolismABSTRACT
The physiological role of immune cells in the regulation of postprandial glucose metabolism has not been fully elucidated. We have found that adipose tissue macrophages produce interleukin-10 (IL-10) upon feeding, which suppresses hepatic glucose production in cooperation with insulin. Both elevated insulin and gut-microbiome-derived lipopolysaccharide in response to feeding are required for IL-10 production via the Akt/mammalian target of rapamycin (mTOR) pathway. Indeed, myeloid-specific knockout of the insulin receptor or bone marrow transplantation of mutant TLR4 marrow cells results in increased expression of gluconeogenic genes and impaired glucose tolerance. Furthermore, myeloid-specific Akt1 and Akt2 knockout results in similar phenotypes that are rescued by additional knockout of TSC2, an inhibitor of mTOR. In obesity, IL-10 production is impaired due to insulin resistance in macrophages, whereas adenovirus-mediated expression of IL-10 ameliorates postprandial hyperglycemia. Thus, the orchestrated response of the endogenous hormone and gut environment to feeding is a key regulator of postprandial glycemia.
Subject(s)
Adipose Tissue/drug effects , Hyperglycemia/pathology , Insulin/pharmacology , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Proto-Oncogene Proteins c-akt/physiology , TOR Serine-Threonine Kinases/metabolism , Adipose Tissue/metabolism , Animals , Blood Glucose/analysis , Gluconeogenesis/genetics , Hyperglycemia/etiology , Hyperglycemia/metabolism , Hypoglycemic Agents/pharmacology , Insulin Resistance , Interleukin-10/physiology , Macrophages/metabolism , Male , Mice , Mice, Inbred C3H , Mice, Knockout , Postprandial Period , Signal Transduction , TOR Serine-Threonine Kinases/genetics , Tuberous Sclerosis Complex 2 Protein/physiologyABSTRACT
Dysregulation of early neurodevelopment is implicated in macrocephaly/autism disorders. However, the mechanism underlying this dysregulation, particularly in human cells, remains poorly understood. Mutations in the small GTPase gene RAB39b are associated with X-linked macrocephaly, autism spectrum disorder (ASD), and intellectual disability. The in vivo roles of RAB39b in the brain remain unknown. We generated Rab39b knockout (KO) mice and found that they exhibited cortical neurogenesis impairment, macrocephaly, and hallmark ASD behaviors, which resembled patient phenotypes. We also produced mutant human cerebral organoids that were substantially enlarged due to the overproliferation and impaired differentiation of neural progenitor cells (NPCs), which resemble neurodevelopmental deficits in KO mice. Mechanistic studies reveal that RAB39b interacts with PI3K components and its deletion promotes PI3K-AKT-mTOR signaling in NPCs of mouse cortex and cerebral organoids. The mTOR activity is robustly enhanced in mutant outer radial glia cells (oRGs), a subtype of NPCs barely detectable in rodents but abundant in human brains. Inhibition of AKT signaling rescued enlarged organoid sizes and NPC overproliferation caused by RAB39b mutations. Therefore, RAB39b mutation promotes PI3K-AKT-mTOR activity and alters cortical neurogenesis, leading to macrocephaly and autistic-like behaviors. Our studies provide new insights into neurodevelopmental dysregulation and common pathways associated with ASD across species.
Subject(s)
Autistic Disorder/genetics , Cerebral Cortex/embryology , Megalencephaly/genetics , Neurogenesis/genetics , rab GTP-Binding Proteins/genetics , Animals , Autistic Disorder/physiopathology , Behavior, Animal/physiology , Cell Differentiation/genetics , Cell Proliferation/genetics , Cerebral Cortex/cytology , Gene Deletion , Humans , Megalencephaly/physiopathology , Mice , Mice, Knockout , Models, Animal , Organoids/cytology , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/genetics , Stem Cells/cytology , TOR Serine-Threonine Kinases/metabolism , rab GTP-Binding Proteins/metabolismABSTRACT
The AKT-mTOR pathway is a central regulator of cell growth and metabolism. Upon sustained mTOR activity, AKT activity is attenuated by a feedback loop that restrains upstream signaling. However, how cells control the signals that limit AKT activity is not fully understood. Here, we show that MASTL/Greatwall, a cell cycle kinase that supports mitosis by phosphorylating the PP2A/B55 inhibitors ENSA/ARPP19, inhibits PI3K-AKT activity by sustaining mTORC1- and S6K1-dependent phosphorylation of IRS1 and GRB10. Genetic depletion of MASTL results in an inefficient feedback loop and AKT hyperactivity. These defects are rescued by the expression of phosphomimetic ENSA/ARPP19 or inhibition of PP2A/B55 phosphatases. MASTL is directly phosphorylated by mTORC1, thereby limiting the PP2A/B55-dependent dephosphorylation of IRS1 and GRB10 downstream of mTORC1. Downregulation of MASTL results in increased glucose uptake in vitro and increased glucose tolerance in adult mice, suggesting the relevance of the MASTL-PP2A/B55 kinase-phosphatase module in controlling AKT and maintaining metabolic homeostasis.
Subject(s)
Mechanistic Target of Rapamycin Complex 1 , Protein Phosphatase 2 , Protein Serine-Threonine Kinases , Animals , Mice , Cell Cycle/genetics , Glucose/metabolism , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Mitosis , Phosphatidylinositol 3-Kinases/genetics , Phosphorylation , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
The maintenance of sodium/potassium (Na+ /K+ ) homeostasis in plant cells is essential for salt tolerance. Plants export excess Na+ out of cells mainly through the Salt Overly Sensitive (SOS) pathway, activated by a calcium signal; however, it is unknown whether other signals regulate the SOS pathway and how K+ uptake is regulated under salt stress. Phosphatidic acid (PA) is emerging as a lipid signaling molecule that modulates cellular processes in development and the response to stimuli. Here, we show that PA binds to the residue Lys57 in SOS2, a core member of the SOS pathway, under salt stress, promoting the activity and plasma membrane localization of SOS2, which activates the Na+ /H+ antiporter SOS1 to promote the Na+ efflux. In addition, we reveal that PA promotes the phosphorylation of SOS3-like calcium-binding protein 8 (SCaBP8) by SOS2 under salt stress, which attenuates the SCaBP8-mediated inhibition of Arabidopsis K+ transporter 1 (AKT1), an inward-rectifying K+ channel. These findings suggest that PA regulates the SOS pathway and AKT1 activity under salt stress, promoting Na+ efflux and K+ influx to maintain Na+ /K+ homeostasis.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Protein Serine-Threonine Kinases , Salt Stress , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Homeostasis , Phosphatidic Acids/metabolism , Potassium/metabolism , Protein Serine-Threonine Kinases/metabolism , Salt Stress/genetics , Sodium/metabolismABSTRACT
The dorsal aorta (DA) is the first major blood vessel to develop in the embryonic cardiovascular system. Its formation is governed by a coordinated process involving the migration, specification, and arrangement of angioblasts into arterial and venous lineages, a process conserved across species. Although vascular endothelial growth factor a (VEGF-A) is known to drive DA specification and formation, the kinases involved in this process remain ambiguous. Thus, we investigated the role of protein kinase B (Akt) in zebrafish by generating a quadruple mutant (aktΔ/Δ), in which expression and activity of all Akt genes - akt1, -2, -3a and -3b - are strongly decreased. Live imaging of developing aktΔ/Δ DA uncovers early arteriovenous malformations. Single-cell RNA-sequencing analysis of aktΔ/Δ endothelial cells corroborates the impairment of arterial, yet not venous, cell specification. Notably, endothelial specific expression of ligand-independent activation of Notch or constitutively active Akt1 were sufficient to re-establish normal arterial specification in aktΔ/Δ. The Akt loss-of-function mutant unveils that Akt kinase can act upstream of Notch in arterial endothelial cells, and is involved in proper embryonic artery specification. This sheds light on cardiovascular development, revealing a mechanism behind congenital malformations.
Subject(s)
Arteries , Proto-Oncogene Proteins c-akt , Receptors, Notch , Zebrafish Proteins , Zebrafish , Animals , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/genetics , Zebrafish Proteins/metabolism , Zebrafish Proteins/genetics , Receptors, Notch/metabolism , Receptors, Notch/genetics , Arteries/embryology , Arteries/metabolism , Gene Expression Regulation, Developmental , Endothelial Cells/metabolism , Signal Transduction , Mutation/genetics , Embryo, Nonmammalian/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3ß (GSK3ß) at mitochondria-endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK-I) to the voltage-dependent anion channel (VDAC) on mitochondria. Binding of HK-I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon-γ (IFN-γ) in memory T cells. Subcellular organization of mTORC2-AKT-GSK3ß at mitochondria-ER contact sites, promoting HK-I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Endoplasmic Reticulum/metabolism , Energy Metabolism , Immunologic Memory , Mitochondria/metabolism , Signal Transduction , Cell Respiration , Endoplasmic Reticulum/ultrastructure , Glycogen Synthase Kinase 3 beta/metabolism , Glycolysis , Intracellular Membranes/metabolism , Lymphocyte Activation , Mechanistic Target of Rapamycin Complex 2/metabolism , Mitochondria/ultrastructure , Models, Biological , Proto-Oncogene Proteins c-akt/metabolism , Rapamycin-Insensitive Companion of mTOR Protein/deficiencyABSTRACT
Positive selection of germinal center (GC) B cells is driven by B cell receptor (BCR) affinity and requires help from follicular T helper cells. The transcription factors c-Myc and Foxo1 are critical for GC B cell selection and survival. However, how different affinity-related signaling events control these transcription factors in a manner that links to selection is unknown. Here we showed that GC B cells reprogram CD40 and BCR signaling to transduce via NF-κB and Foxo1, respectively, whereas naive B cells propagate both signals downstream of either receptor. Although either BCR or CD40 ligation induced c-Myc in naive B cells, both signals were required to highly induce c-Myc, a critical mediator of GC B cell survival and cell cycle reentry. Thus, GC B cells rewire their signaling to enhance selection stringency via a requirement for both antigen receptor- and T cell-mediated signals to induce mediators of positive selection.
Subject(s)
CD40 Antigens/physiology , Germinal Center/immunology , Proto-Oncogene Proteins c-myc/biosynthesis , Receptors, Antigen, B-Cell/physiology , Signal Transduction/physiology , Animals , Forkhead Box Protein O1/physiology , MAP Kinase Signaling System/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , NF-kappa B/physiology , Phosphatidylinositol 3-Kinases/physiology , Proto-Oncogene Proteins c-akt/physiology , Syk Kinase/physiologyABSTRACT
The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.
Subject(s)
DNA Topoisomerases, Type II/genetics , Herpesvirus 1, Human/genetics , Nuclear Proteins/genetics , Proto-Oncogene Proteins c-akt/genetics , Virus Latency/genetics , Animals , DNA Breaks, Double-Stranded , DNA Damage/genetics , DNA End-Joining Repair/genetics , DNA Repair/genetics , DNA Repair Enzymes/genetics , DNA-Binding Proteins/genetics , Herpesvirus 1, Human/pathogenicity , Humans , MRE11 Homologue Protein/genetics , Mechanistic Target of Rapamycin Complex 1/genetics , Neurons/metabolism , Neurons/virology , Phosphorylation , Rats , Signal Transduction/genetics , TOR Serine-Threonine Kinases/geneticsABSTRACT
Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.
Subject(s)
Breast Neoplasms , Cell Nucleus , Poly (ADP-Ribose) Polymerase-1 , Proto-Oncogene Proteins c-akt , Humans , Breast Neoplasms/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , Cell Nucleus/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/genetics , Cell Line, Tumor , PTEN Phosphohydrolase/metabolism , PTEN Phosphohydrolase/genetics , Amino Acyl-tRNA Synthetases/metabolism , Amino Acyl-tRNA Synthetases/genetics , Active Transport, Cell Nucleus , Nuclear Localization Signals/metabolismABSTRACT
The Shoc2 scaffold protein is crucial in transmitting signals within the Epidermal Growth Factor Receptor (EGFR)-mediated Extracellular signal-Regulated Kinase (ERK1/2) pathway. While the significance of Shoc2 in this pathway is well-established, the precise mechanisms through which Shoc2 governs signal transmission remain to be fully elucidated. Hereditary variants in Shoc2 are responsible for Noonan Syndrome with Loose anagen Hair (NSLH). However, due to the absence of known enzymatic activity in Shoc2, directly assessing how these variants affect its function is challenging. ERK1/2 phosphorylation is used as a primary parameter of Shoc2 function, but the impact of Shoc2 mutants on the pathway activation is unclear. This study investigates how the NSLH-associated Shoc2 variants influence EGFR signals in the context of the ERK1/2 and AKT downstream signaling pathways. We show that when the ERK1/2 pathway is a primary signaling pathway activated downstream of EGFR, Shoc2 variants cannot upregulate ERK1/2 phosphorylation to the level of the WT Shoc2. Yet, when the AKT and ERK1/2 pathways were activated, in cells expressing Shoc2 variants, ERK1/2 phosphorylation was higher than in cells expressing WT Shoc2. In cells expressing the Shoc2 NSLH mutants, we found that the AKT signaling pathway triggers the PAK activation, followed by phosphorylation of Raf-1/MEK1/2 and activation of the ERK1/2 signaling axis. Hence, our studies reveal a previously unrecognized feedback regulation downstream of the EGFR and provide additional evidence for the role of Shoc2 as a "gatekeeper" in controlling the selection of downstream effectors within the EGFR signaling network.
Subject(s)
ErbB Receptors , MAP Kinase Signaling System , Proto-Oncogene Proteins c-akt , Humans , ErbB Receptors/metabolism , ErbB Receptors/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/genetics , MAP Kinase Signaling System/genetics , Phosphorylation , Noonan Syndrome/genetics , Noonan Syndrome/metabolism , Signal Transduction/genetics , Son of Sevenless Proteins/metabolism , Son of Sevenless Proteins/genetics , Mutation , HEK293 Cells , Intracellular Signaling Peptides and Proteins , Mitogen-Activated Protein Kinase 3ABSTRACT
Interferon regulatory factor 3 (IRF3)-induced type I interferon (I-IFN) production plays key roles in both antiviral and autoimmune responses. IRF3 phosphorylation, dimerization, and nuclear localization are needed for its activation and function, but the precise regulatory mechanisms remain to be explored. Here, we show that the serine/threonine kinase AKT2 interacts with IRF3 and phosphorylates it on Thr207, thereby attenuating IRF3 nuclear translocation in a 14-3-3ε-dependent manner and reducing I-IFN production. We further find that AKT2 expression is downregulated in viral-infected macrophages or in monocytes and tissue samples from systemic lupus erythematosus (SLE) patients and mouse models. Akt2-deficient mice exhibit increased I-IFN induction and reduced mortality in response to viral infection, but aggravated severity of SLE. Overexpression of AKT2 kinase-inactive or IRF3-T207A mutants in zebrafish supports that AKT2 negatively regulates I-IFN production and antiviral response in a kinase-dependent manner. This negative role of AKT2 in IRF3-induced I-IFN production suggests that AKT2 may be therapeutically targeted to differentially regulate antiviral infection and SLE.
Subject(s)
Interferon-beta/biosynthesis , Lupus Erythematosus, Systemic , Zebrafish , Animals , Antiviral Agents , Humans , Lupus Erythematosus, Systemic/genetics , Mice , Phosphorylation , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Zebrafish/metabolismABSTRACT
Hemochorial placentation involves the differentiation of invasive trophoblast cells, specialized cells that possess the capacity to exit the placenta and invade into the uterus where they restructure the vasculature. Invasive trophoblast cells arise from a well-defined compartment within the placenta, referred to as the junctional zone in rat and the extravillous trophoblast cell column in human. In this study, we investigated roles for AKT1, a serine/threonine kinase, in placental development using a genome-edited/loss-of-function rat model. Disruption of AKT1 resulted in placental, fetal and postnatal growth restriction. Forkhead box O4 (Foxo4), which encodes a transcription factor and known AKT substrate, was abundantly expressed in the junctional zone and in invasive trophoblast cells of the rat placentation site. Foxo4 gene disruption using genome editing resulted in placentomegaly, including an enlarged junctional zone. AKT1 and FOXO4 regulate the expression of many of the same transcripts expressed by trophoblast cells, but in opposite directions. In summary, we have identified AKT1 and FOXO4 as part of a regulatory network that reciprocally controls critical indices of hemochorial placenta development.
Subject(s)
Placenta , Placentation , Animals , Female , Pregnancy , Rats , Cell Cycle Proteins/metabolism , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation , Placenta/metabolism , Placentation/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Trophoblasts , UterusABSTRACT
Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17â¼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.
Subject(s)
Colitis/immunology , Phosphatidylinositol 3-Kinases/immunology , Repressor Proteins/immunology , TOR Serine-Threonine Kinases/immunology , Ubiquitin-Protein Ligases/immunology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cell Differentiation , Colitis/genetics , Colitis/pathology , Disease Models, Animal , Female , Forkhead Box Protein O1/genetics , Forkhead Box Protein O1/immunology , Gene Expression Regulation , Germinal Center/immunology , Germinal Center/pathology , Interleukin-2 Receptor alpha Subunit/genetics , Interleukin-2 Receptor alpha Subunit/immunology , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , MicroRNAs/genetics , MicroRNAs/immunology , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/immunology , Phosphatidylinositol 3-Kinases/genetics , Primary Cell Culture , Repressor Proteins/deficiency , Repressor Proteins/genetics , Signal Transduction , Spleen/immunology , Spleen/pathology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , TOR Serine-Threonine Kinases/genetics , Th17 Cells/immunology , Th17 Cells/pathology , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/geneticsABSTRACT
ATP2B1 is a known regulator of calcium (Ca2+) cellular export and homeostasis. Diminished levels of intracellular Ca2+ content have been suggested to impair SARS-CoV-2 replication. Here, we demonstrate that a nontoxic caloxin-derivative compound (PI-7) reduces intracellular Ca2+ levels and impairs SARS-CoV-2 infection. Furthermore, a rare homozygous intronic variant of ATP2B1 is shown to be associated with the severity of COVID-19. The mechanism of action during SARS-CoV-2 infection involves the PI3K/Akt signaling pathway activation, inactivation of FOXO3 transcription factor function, and subsequent transcriptional inhibition of the membrane and reticulum Ca2+ pumps ATP2B1 and ATP2A1, respectively. The pharmacological action of compound PI-7 on sustaining both ATP2B1 and ATP2A1 expression reduces the intracellular cytoplasmic Ca2+ pool and thus negatively influences SARS-CoV-2 replication and propagation. As compound PI-7 lacks toxicity in vitro, its prophylactic use as a therapeutic agent against COVID-19 is envisioned here.