RESUMEN
Many genetic studies have established the kinase activity of inositol phosphate multikinase (IPMK) is required for the synthesis of higher-order inositol phosphate signaling molecules, the regulation of gene expression and control of the cell cycle. These genetic studies await orthogonal validation by specific IPMK inhibitors, but no such inhibitors have been synthesized. Here, we report complete chemical synthesis, cellular characterization, structure-activity relationships and rodent pharmacokinetics of a novel series of highly potent IPMK inhibitors. The first-generation compound 1 (UNC7437) decreased cellular proliferation and tritiated inositol phosphate levels in metabolically labeled human U251-MG glioblastoma cells. Compound 1 also regulated the transcriptome of these cells, selectively regulating genes that are enriched in cancer, inflammatory and viral infection pathways. Further optimization of compound 1 eventually led to compound 15 (UNC9750), which showed improved potency and pharmacokinetics in rodents. Compound 15 specifically inhibited cellular accumulation of InsP 5 , a direct product of IPMK kinase activity, while having no effect on InsP 6 levels, revealing a novel metabolic signature detected for the first time by rapid chemical attenuation of cellular IPMK activity. These studies designed, optimized and synthesized a new series of IPMK inhibitors, which reduces glioblastoma cell growth, induces a novel InsP 5 metabolic signature, and reveals novel aspects inositol phosphate cellular metabolism and signaling.
RESUMEN
Mechanistic Target of Rapamycin (mTOR) binds the small metabolite inositol hexakisphosphate (IP6) as shown in structures of mTOR, however it remains unclear if IP6, or any other inositol phosphate species, can activate mTOR kinase activity. Here, we show that multiple, exogenously added inositol phosphate species (IP6, IP5, IP4 and IP3) can all enhance the ability of mTOR and mTORC1 to auto-phosphorylate and incorporate radiolabeled phosphate into peptide substrates in in vitro kinase reactions. Although IP6 did not affect the apparent KM of mTORC1 for ATP, monitoring kinase activity over longer reaction times showed increased product formation, suggesting inositol phosphates stabilize an active form of mTORC1 in vitro. The effects of IP6 on mTOR were reversible, suggesting IP6 bound to mTOR can be exchanged dynamically with the free solvent. Interestingly, we also observed that IP6 could alter mTOR solubility and electrophoretic mobility in SDS-PAGE in the presence of manganese, suggesting divalent cations may play a role in inositol phosphate regulation of mTOR. Together, these data suggest for the first time that multiple inositol phosphate species (IP4, IP5 and IP6) can dynamically regulate mTOR and mTORC1 by promoting a stable, active state of the kinase. Our data suggest that studies of the dynamics of inositol phosphate regulation of mTOR are well justified.
RESUMEN
Histone deacetylases (HDACs) repress transcription by catalyzing the removal of acetyl groups from histones. Class 1 HDACs are activated by inositol phosphate signaling molecules in vitro , but it is unclear if this regulation occurs in human cells. Inositol Polyphosphate Multikinase (IPMK) is required for production of inositol hexakisphosphate (IP6), pentakisphosphate (IP5) and certain tetrakisphosphate (IP4) species, all known activators of Class 1 HDACs in vitro . Here, we generated IPMK knockout (IKO) human U251 glioblastoma cells, which decreased cellular inositol phosphate levels and increased histone H4-acetylation by mass spectrometry. ChIP-seq showed IKO increased H4-acetylation at IKO-upregulated genes, but H4-acetylation was unchanged at IKO-downregulated genes, suggesting gene-specific responses to IPMK knockout. HDAC deacetylase enzyme activity was decreased in HDAC3 immunoprecipitates from IKO vs . wild-type cells, while deacetylase activity of other Class 1 HDACs had no detectable changes in activity. Wild-type IPMK expression in IKO cells fully rescued HDAC3 deacetylase activity, while kinase-dead IPMK expression had no effect. Further, the deficiency in HDAC3 activity in immunoprecipitates from IKO cells could be fully rescued by addition of synthesized IP4 (Ins(1,4,5,6)P4) to the enzyme assay, while control inositol had no effect. These data suggest that cellular IPMK-dependent inositol phosphates are required for full HDAC3 enzyme activity and proper histone H4-acetylation. Implications for targeting IPMK in HDAC3-dependent diseases are discussed.
RESUMEN
The accidental discovery of PI5P (phosphatidylinositol-5-phosphate) was published 25 years ago, when PIP5K type II (phosphoinositide-4-phosphate 5-kinase) was shown to actually be a 4-kinase that uses PI5P as a substrate to generate PI(4,5)P2. Consequently, PIP5K type II was renamed to PI5P4K, or PIP4K for short, and PI5P became the last of the 7 signaling phosphoinositides to be discovered. Much of what we know about PI5P comes from genetic studies of PIP4K, as the pathways for PI5P synthesis, the downstream targets of PI5P and how PI5P affects cellular function all remain largely enigmatic. Nevertheless, PI5P and PI5P-dependent PI(4,5)P2 synthesis have been clearly implicated in metabolic homeostasis and in diseases such as cancer. Here, we review the past 25 years of PI5P research, with particular emphasis on the impact this small signaling lipid has on human health.
RESUMEN
Transporters of the inner mitochondrial membrane are essential to metabolism. We demonstrate that metabolism as represented by expression of genes encoding SLC25 transporters differentiates human cancers. Tumor to normal tissue expression ratios for clear cell renal cell carcinoma, colon adenocarcinoma, lung adenocarcinoma and breast invasive carcinoma were found to be highly significant. Affinity propagation trained on SLC25 gene expression patterns from 19 human cancer types (6825 TCGA samples) and normal tissues (2322 GTEx samples) was used to generate clusters. They differentiate cancers from normal tissues. They also indicate cancer subtypes with survivals distinct from the total patient population of the cancer type. Probing the kidney, colon, lung, and breast cancer clusters, subtype pairs of cancers were identified with distinct prognoses and differing in expression of protein coding genes from among 2080 metabolic enzymes assayed. We demonstrate that SLC25 expression clusters facilitate the identification of the tissue-of-origin, essential to efficacy of most cancer therapies, of CUPs (cancer-unknown-primary) known to have poor prognoses. Different cancer types within a single cluster have similar metabolic patterns and this raises the possibility that such cancers may respond similarly to existing and new anti-cancer therapies.
Asunto(s)
Adenocarcinoma , Neoplasias de la Mama , Carcinoma de Células Renales , Neoplasias del Colon , Neoplasias Renales , Adenocarcinoma/genética , Neoplasias de la Mama/genética , Carcinoma de Células Renales/patología , Neoplasias del Colon/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Renales/patología , PronósticoRESUMEN
Chronic exposure of pancreatic ß-cells to excess glucose can lead to metabolic acceleration and loss of stimulus-secretion coupling. Here, we examined how exposure to excess glucose (defined here as concentrations above 5 mM) affects mTORC1 signaling and the metabolism of ß-cells. Acute exposure to excess glucose stimulated glycolysis-dependent mTORC1 signaling, without changes in the PI3K or AMPK pathways. Prolonged exposure to excess glucose led to hyperactivation of mTORC1 and metabolic acceleration, characterized by higher basal respiration and maximal respiratory capacity, increased energy demand, and enhanced flux through mitochondrial pyruvate metabolism. Inhibition of pyruvate transport to the mitochondria decelerated the metabolism of ß-cells chronically exposed to excess glucose and re-established glucose-dependent mTORC1 signaling, disrupting a positive feedback loop for mTORC1 hyperactivation. mTOR inhibition had positive and negative impacts on various metabolic pathways and insulin secretion, demonstrating a role for mTOR signaling in the long-term metabolic adaptation of ß-cells to excess glucose.
RESUMEN
Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.
Asunto(s)
Camptotecina/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Resistencia a Antineoplásicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Topoisomerasa I/farmacología , Ubiquitina/metabolismo , Proteína BRCA1/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Edición Génica , Humanos , Autoantígeno Ku/metabolismo , Complejos Multiproteicos/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosforilación , Unión Proteica , Proteína Quinasa C/metabolismo , Proteolisis , Interferencia de ARNRESUMEN
Despite being one of the most studied signalling pathways, precisely how phospholipid synthesis is regulated in the phosphoinositide signalling cascade remains unclear. The scaffold protein IQGAP1 is now shown to orchestrate the assembly of a multi-enzyme complex that streamlines PtdIns(3,4,5)P3 synthesis to facilitate Akt activation in response to extracellular stimuli.
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Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transducción de Señal , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Humanos , Modelos Biológicos , Neoplasias/metabolismo , FosforilaciónRESUMEN
Type 2 diabetes is a complex disease. It results from a failure of the body to maintain energy homoeostasis. Multicellular organisms have evolved complex strategies to preserve a relatively stable internal nutrient environment, despite fluctuations in external nutrient availability. This complex strategy involves the co-ordinated responses of multiple organs to promote storage or mobilization of energy sources according to the availability of nutrients and cellular bioenergetics needs. The endocrine pancreas plays a central role in these processes by secreting insulin and glucagon. When this co-ordinated effort fails, hyperglycaemia and hyperlipidaemia develops, characterizing a state of metabolic imbalance and ultimately overt diabetes. Although diabetes is most likely a collection of diseases, scientists are starting to identify genetic components and environmental triggers. Genome-wide association studies revealed that by and large, gene variants associated with type 2 diabetes are implicated in pancreatic ß-cell function, suggesting that the ß-cell may be the weakest link in the chain of events that results in diabetes. Thus, it is critical to understand how environmental cues affect the ß-cell. Phosphoinositides are important 'decoders' of environmental cues. As such, these lipids have been implicated in cellular responses to a wide range of growth factors, hormones, stress agents, nutrients and metabolites. Here we will review some of the well-established and potential new roles for phosphoinositides in ß-cell function/dysfunction and discuss how our knowledge of phosphoinositide signalling could aid in the identification of potential strategies for treating or preventing type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Fosfatidilinositoles/metabolismo , Transducción de Señal , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Animales , Humanos , Insulina/metabolismo , Secreción de InsulinaRESUMEN
Phosphoinositides play critical roles in the transduction of extracellular signals through the plasma membrane and also in endomembrane events important for vesicle trafficking and organelle function (Di Paolo and De Camilli, Nature 443(7112):651-657, 2006). The response triggered by these lipids is heavily dependent on the microenvironment in which they are found. HPLC analysis of labeled phosphoinositides allows quantification of the levels of each phosphoinositide species relative to their precursor, phosphatidylinositol. When combined with subcellular fractionation techniques, this strategy allows measurement of the relative phosphoinositide composition of each membrane fraction or organelle and determination of the microenvironment in which each species is enriched. Here, we describe the steps to separate and quantify total or localized phosphoinositides from cultured cells.
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Fraccionamiento Celular/métodos , Membrana Celular , Fosfatidilinositoles , Membrana Celular/química , Cromatografía Líquida de Alta Presión , Fosfatidilinositoles/química , Coloración y Etiquetado , Fracciones Subcelulares , Tritio/químicaRESUMEN
In this issue of Cancer Cell, Ooms and colleagues show that the lipid phosphatase PIPP/INPP5J, frequently inactivated in triple-negative breast cancers, functions as a tumor suppressor by specifically modulating the activity of AKT1 in the context of oncogenic PI3K signaling, leading to inhibition of metastatic dissemination.
Asunto(s)
Neoplasias de la Mama/genética , Proliferación Celular/genética , Monoéster Fosfórico Hidrolasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Animales , HumanosRESUMEN
Phosphatidylinositol-5-phosphate 4-kinases (PIP4ks) are a family of lipid kinases that specifically use phosphatidylinositol 5-monophosphate (PI-5-P) as a substrate to synthesize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Suppression of PIP4k function in Drosophila results in smaller cells and reduced target of rapamycin complex 1 (TORC1) signaling. We showed that the γ isoform of PIP4k stimulated signaling through mammalian TORC1 (mTORC1). Knockdown of PIP4kγ reduced cell mass in cells in which mTORC1 is constitutively activated by Tsc2 deficiency. In Tsc2 null cells, mTORC1 activation was partially independent of amino acids or glucose and glutamine. PIP4kγ knockdown inhibited the nutrient-independent activation of mTORC1 in Tsc2 knockdown cells and reduced basal mTORC1 signaling in wild-type cells. PIP4kγ was phosphorylated by mTORC1 and associated with the complex. Phosphorylated PIP4kγ was enriched in light microsomal vesicles, whereas the unphosphorylated form was enriched in heavy microsomal vesicles associated with the Golgi. Furthermore, basal mTORC1 signaling was enhanced by overexpression of unphosphorylated wild-type PIP4kγ or a phosphorylation-defective mutant and decreased by overexpression of a phosphorylation-mimetic mutant. Together, these results demonstrate that PIP4kγ and mTORC1 interact in a self-regulated feedback loop to maintain low and tightly regulated mTORC1 activation during starvation.
Asunto(s)
Complejos Multiproteicos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Citoplasma/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Mutación , Fosforilación , Transducción de Señal , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.
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1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , Malaria/tratamiento farmacológico , Malaria/parasitología , Plasmodium/efectos de los fármacos , Plasmodium/enzimología , 1-Fosfatidilinositol 4-Quinasa/química , 1-Fosfatidilinositol 4-Quinasa/genética , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Citocinesis/efectos de los fármacos , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Ácidos Grasos/metabolismo , Femenino , Hepatocitos/parasitología , Humanos , Imidazoles/metabolismo , Imidazoles/farmacología , Estadios del Ciclo de Vida/efectos de los fármacos , Macaca mulatta , Masculino , Modelos Biológicos , Modelos Moleculares , Fosfatos de Fosfatidilinositol/metabolismo , Plasmodium/clasificación , Plasmodium/crecimiento & desarrollo , Pirazoles/metabolismo , Pirazoles/farmacología , Quinoxalinas/metabolismo , Quinoxalinas/farmacología , Reproducibilidad de los Resultados , Esquizontes/citología , Esquizontes/efectos de los fármacos , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Here, we show that a subset of breast cancers express high levels of the type 2 phosphatidylinositol-5-phosphate 4-kinases α and/or ß (PI5P4Kα and ß) and provide evidence that these kinases are essential for growth in the absence of p53. Knocking down PI5P4Kα and ß in a breast cancer cell line bearing an amplification of the gene encoding PI5P4K ß and deficient for p53 impaired growth on plastic and in xenografts. This growth phenotype was accompanied by enhanced levels of reactive oxygen species (ROS) leading to senescence. Mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly however, PIP4K2A(-/-), PIP4K2B(+/-), and TP53(-/-) mice were viable and had a dramatic reduction in tumor formation compared to TP53(-/-) littermates. These results indicate that inhibitors of PI5P4Ks could be effective in preventing or treating cancers with mutations in TP53.
Asunto(s)
Neoplasias de la Mama/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Respiración de la Célula , Senescencia Celular , Embrión de Mamíferos/metabolismo , Técnicas de Silenciamiento del Gen , Genes Letales , Xenoinjertos , Humanos , Ratones , Trasplante de Neoplasias , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) that controls cell proliferation, growth, survival, metabolism, and migration by activating the PI3K (phosphatidylinositol 3-kinase)-AKT and ERK (extracellular signal-regulated kinase)-RSK (ribosomal S6 kinase) pathways. EGFR signaling to these pathways is temporally and spatially regulated. Endocytic trafficking controls the access of EGFR to these downstream effectors and also its degradation, which terminates EGFR signaling. We showed that AKT facilitated the endocytic trafficking of EGFR to promote its degradation. Interfering with AKT signaling reduced both EGFR recycling and the rate of EGFR degradation. In AKT-impaired cells, EGFRs were unable to reach the cell surface or the lysosomal compartment and accumulated in the early endosomes, resulting in prolonged signaling and increased activation of ERK and RSK. Upon EGF stimulation, AKT phosphorylated and activated the kinase PIKfyve [FYVE-containing phosphatidylinositol 3-phosphate 5-kinase], which promoted vesicle trafficking to lysosomes. PIKfyve activation promoted EGFR degradation. Similar regulation occurred with platelet-derived growth factor receptor (PDGFR), suggesting that AKT phosphorylation and activation of PIKfyve is likely to be a common feedback mechanism for terminating RTK signaling and reducing receptor abundance.
Asunto(s)
Receptores ErbB/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Western Blotting , Línea Celular , Endocitosis/efectos de los fármacos , Endosomas/metabolismo , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Células HEK293 , Humanos , Lisosomas/metabolismo , Microscopía Confocal , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/genética , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Transducción de Señal/efectos de los fármacosRESUMEN
Although phosphatidylinositol 5-phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3-kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P-binding motifs, we identified the phosphoinositide 5-kinase PIKfyve and the phosphoinositide 3-phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P(2). The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P-producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.
Asunto(s)
Movimiento Celular/fisiología , Drosophila melanogaster/metabolismo , Fibroblastos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/biosíntesis , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Animales , Sitios de Unión , Línea Celular , Fosfatidilinositol 3-Quinasas Clase III/genética , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Drosophila melanogaster/genética , Fibroblastos/citología , Regulación de la Expresión Génica , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Tirosina Fosfatasas no Receptoras/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas no Receptoras/genética , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
Decremental loss of PTEN results in cancer susceptibility and tumor progression. PTEN elevation might therefore be an attractive option for cancer prevention and therapy. We have generated several transgenic mouse lines with PTEN expression elevated to varying levels by taking advantage of bacterial artificial chromosome (BAC)-mediated transgenesis. The "Super-PTEN" mutants are viable and show reduced body size due to decreased cell number, with no effect on cell size. Unexpectedly, PTEN elevation at the organism level results in healthy metabolism characterized by increased energy expenditure and reduced body fat accumulation. Cells derived from these mice show reduced glucose and glutamine uptake and increased mitochondrial oxidative phosphorylation and are resistant to oncogenic transformation. Mechanistically we find that PTEN elevation orchestrates this metabolic switch by regulating PI3K-dependent and -independent pathways and negatively impacting two of the most pronounced metabolic features of tumor cells: glutaminolysis and the Warburg effect.
Asunto(s)
Fosfohidrolasa PTEN/metabolismo , Transducción de Señal , Animales , Tamaño Corporal , Recuento de Células , Proliferación Celular , Respiración de la Célula , Metabolismo Energético , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Phagocytosis is a pivotal process by which macrophages eliminate microorganisms after recognition by pathogen sensors. Here we unexpectedly found that the self ligand and cell surface receptor SLAM functioned not only as a costimulatory molecule but also as a microbial sensor that controlled the killing of gram-negative bacteria by macrophages. SLAM regulated activity of the NADPH oxidase NOX2 complex and phagolysosomal maturation after entering the phagosome, following interaction with the bacterial outer membrane proteins OmpC and OmpF. SLAM recruited a complex containing the intracellular class III phosphatidylinositol kinase Vps34, its regulatory protein kinase Vps15 and the autophagy-associated molecule beclin-1 to the phagosome, which was responsible for inducing the accumulation of phosphatidylinositol-3-phosphate, a regulator of both NOX2 function and phagosomal or endosomal fusion. Thus, SLAM connects the gram-negative bacterial phagosome to ubiquitous cellular machinery responsible for the control of bacterial killing.
Asunto(s)
Antígenos CD/metabolismo , Infecciones por Escherichia coli/inmunología , Escherichia coli/inmunología , Macrófagos/inmunología , Fagosomas/inmunología , Receptores de Superficie Celular/metabolismo , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Animales , Antígenos CD/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Bacterianas/genética , Beclina-1 , Células Cultivadas , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Macrófagos/microbiología , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Chaperonas Moleculares/genética , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Fagocitosis , Fagosomas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Porinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/genética , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Proteína de Clasificación Vacuolar VPS15RESUMEN
Vacuolar protein sorting 34 (Vps34) complexes, the class III PtdIns3 kinase, specifically phosphorylate the D3 position of PtdIns to produce PtdIns3P. Vps34 is involved in the control of multiple key intracellular membrane trafficking pathways including endocytic sorting and autophagy. In mammalian cells, Vps34 interacts with Beclin 1, an ortholog of Atg6 in yeast, to regulate the production of PtdIns3P and autophagy. We show that Vps34 is phosphorylated on Thr159 by Cdk1, which negatively regulates its interaction with Beclin 1 during mitosis. Cdk5/p25, a neuronal Cdk shown to play a role in Alzheimer's disease, can also phosphorylate Thr159 of Vps34. Phosphorylation of Vps34 on Thr159 inhibits its interaction with Beclin 1. We propose that phosphorylation of Thr159 in Vps34 is a key regulatory mechanism that controls the class III PtdIns3 kinase activity in cell-cycle progression, development, and human diseases including neurodegeneration and cancers.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Células HeLa , Humanos , Mitosis , Inhibidores de las Quinasa Fosfoinosítidos-3 , FosforilaciónRESUMEN
PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor that antagonizes signaling through the phosphatidylinositol 3-kinase-Akt pathway. We have demonstrated that subtle decreases in PTEN abundance can have critical consequences for tumorigenesis. Here, we used a computational approach to identify miR-22, miR-25, and miR-302 as three PTEN-targeting microRNA (miRNA) families found within nine genomic loci. We showed that miR-22 and the miR-106b~25 cluster are aberrantly overexpressed in human prostate cancer, correlate with abundance of the miRNA processing enzyme DICER, and potentiate cellular transformation both in vitro and in vivo. We demonstrated that the intronic miR-106b~25 cluster cooperates with its host gene MCM7 in cellular transformation both in vitro and in vivo, so that the concomitant overexpression of MCM7 and the miRNA cluster triggers prostatic intraepithelial neoplasia in transgenic mice. Therefore, the MCM7 gene locus delivers two simultaneous oncogenic insults when amplified or overexpressed in human cancer. Thus, we have uncovered a proto-oncogenic miRNA-dependent network for PTEN regulation and defined the MCM7 locus as a critical factor in initiating prostate tumorigenesis.