RESUMEN
Phosphoinositide 3-kinases (PI3Ks) catalyse the synthesis of specific members of the family of lipids collectively known as 'phosphoinositides'. These PI3Ks products can in turn modulate activation of many downstream proteins, ultimately regulating several cellular processes. Mammalian cells possess eight PI3Ks which are grouped into three classes based on their structure and substrate specificity. While class I and III PI3Ks have been extensively investigated, our understanding of the three class II members has only improved in most recent years. This chapter will summarise some of the available information on mammalian class II PI3Ks and their physiological roles.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II , Fosfatidilinositol 3-Quinasas , Fosfatidilinositol 3-Quinasas Clase II/genética , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , FosfatidilinositolesRESUMEN
Aging is impacted by interventions across species, often converging on metabolic pathways. Transcription factors regulate longevity yet approaches for their pharmacological modulation to exert geroprotection remain sparse. We show that increased expression of the transcription factor Grainyhead 1 (GRH-1) promotes lifespan and pathogen resistance in Caenorhabditis elegans. A compound screen identifies FDA-approved drugs able to activate human GRHL1 and promote nematodal GRH-1-dependent longevity. GRHL1 activity is regulated by post-translational lysine methylation and the phosphoinositide (PI) 3-kinase C2A. Consistently, nematodal longevity following impairment of the PI 3-kinase or insulin/IGF-1 receptor requires grh-1. In BXD mice, Grhl1 expression is positively correlated with lifespan and insulin sensitivity. In humans, GRHL1 expression positively correlates with insulin receptor signaling and also with lifespan. Fasting blood glucose levels, including in individuals with type 2 diabetes, are negatively correlated with GRHL1 expression. Thereby, GRH-1/GRHL1 is identified as a pharmacologically malleable transcription factor impacting insulin signaling and lifespan.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Fosfatidilinositol 3-Quinasas Clase II/genética , Diabetes Mellitus Tipo 2/genética , Factor I del Crecimiento Similar a la Insulina/genética , Insulina/metabolismo , Longevidad/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Animales Modificados Genéticamente , Glucemia/metabolismo , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Regulación de la Expresión Génica , Humanos , Resistencia a la Insulina , Factor I del Crecimiento Similar a la Insulina/metabolismo , Longevidad/efectos de los fármacos , Metilación , Ratones , Papaverina/farmacología , Proteínas Represoras/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Vorinostat/farmacologíaRESUMEN
Phosphatidylinositol 3-kinases (PI3Ks) are critical regulators of many cellular processes including cell survival, proliferation, migration, cytoskeletal reorganization, and intracellular vesicular trafficking. They are a family of lipid kinases that phosphorylate membrane phosphoinositide lipids at the 3' position of their inositol rings, and in mammals they are divided into three classes. The role of the class III PI3K Vps34 is well-established, but recent evidence suggests the physiological significance of class II PI3K isoforms in vesicular trafficking. This review focuses on the recently discovered functions of the distinct PI3K-C2α and PI3K-C2ß class II PI3K isoforms in clathrin-mediated endocytosis and consequent endosomal signaling, and discusses recently reported data on class II PI3K isoforms in different physiological contexts in comparison with class I and III isoforms.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Vesículas Citoplasmáticas/metabolismo , Endocitosis/fisiología , Espacio Intracelular/metabolismo , Animales , Transporte Biológico/fisiología , Endosomas/metabolismo , Humanos , Isoenzimas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Our knowledge on the expression, regulation and roles of the different phosphoinositide 3-kinases (PI3Ks) in platelet signaling and functions has greatly expanded these last twenty years. Much progress has been made in understanding the roles and regulations of class I PI3Ks which produce the lipid second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3). Selective pharmacological inhibitors and genetic approaches have allowed researchers to generate an impressive amount of data on the role of class I PI3Kα, ß, δ and γ in platelet activation and in thrombosis. Furthermore, platelets do also express two class II PI3Ks (PI3KC2α and PI3KC2ß), thought to generate PtdIns(3,4)P2 and PtdIns3P, and the sole class III PI3K (Vps34), known to synthesize PtdIns3P. Recent studies have started to reveal the importance of PI3KC2α and Vps34 in megakaryocytes and platelets, opening new perspective in our comprehension of platelet biology and thrombosis. In this review, we will summarize previous and recent advances on platelet PI3Ks isoforms. The implication of these kinases and their lipid products in fundamental platelet biological processes and thrombosis will be discussed. Finally, the relevance of developing potential antithrombotic strategies by targeting PI3Ks will be examined.
Asunto(s)
Plaquetas/enzimología , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Trombosis/enzimología , Trombosis/terapia , Animales , Plaquetas/patología , Humanos , Isoenzimas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Trombosis/patologíaRESUMEN
It is widely recognized that noncoding genetic variants play important roles in many human diseases, but there are multiple challenges that hinder the identification of functional disease-associated noncoding variants. The number of noncoding variants can be many times that of coding variants; many of them are not functional but in linkage disequilibrium with the functional ones; different variants can have epistatic effects; different variants can affect the same genes or pathways in different individuals; and some variants are related to each other not by affecting the same gene but by affecting the binding of the same upstream regulator. To overcome these difficulties, we propose a novel analysis framework that considers convergent impacts of different genetic variants on protein binding, which provides multiscale information about disease-associated perturbations of regulatory elements, genes, and pathways. Applying it to our whole-genome sequencing data of 918 short-segment Hirschsprung disease patients and matched controls, we identify various novel genes not detected by standard single-variant and region-based tests, functionally centering on neural crest migration and development. Our framework also identifies upstream regulators whose binding is influenced by the noncoding variants. Using human neural crest cells, we confirm cell stage-specific regulatory roles of three top novel regulatory elements on our list, respectively in the RET, RASGEF1A, and PIK3C2B loci. In the PIK3C2B regulatory element, we further show that a noncoding variant found only in the patients affects the binding of the gliogenesis regulator NFIA, with a corresponding up-regulation of multiple genes in the same topologically associating domain.
Asunto(s)
Elementos de Facilitación Genéticos , Enfermedad de Hirschsprung/genética , Regiones Promotoras Genéticas , Fosfatidilinositol 3-Quinasas Clase II/genética , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Variación Genética , Humanos , Intrones , Factores de Transcripción NFI/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Secuenciación Completa del Genoma , Factores de Intercambio de Guanina Nucleótido ras/genéticaRESUMEN
Class II phosphatidylinositol 3-kinases (PI3K), PI3K-C2α and PI3K-C2ß, are involved in cellular processes including endocytosis, cilia formation and autophagy. However, the role of PI3K-C2α and PI3K-C2ß at the organismal level is not well understood. We found that double knockout (KO) mice with both smooth muscle-specific KO of PI3K-C2α and global PI3K-C2ß KO, but not single KO mice of either PI3K-C2α or PI3K-C2ß, exhibited reductions in arterial blood pressure and substantial attenuation of contractile responses of isolated aortic rings. In wild-type vascular smooth muscle cells, double knockdown of PI3K-C2α and PI3K-C2ß but not single knockdown of either PI3K markedly inhibited contraction with reduced phosphorylation of 20-kDa myosin light chain and MYPT1 and Rho activation, but without inhibition of the intracellular Ca2+ mobilization. These data indicate that PI3K-C2α and PI3K-C2ß play the redundant but essential role for vascular smooth muscle contraction and blood pressure regulation mainly through their involvement in Rho activation.
Asunto(s)
Calcio/metabolismo , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Proteínas de Unión al GTP rho/metabolismo , Animales , Presión Sanguínea/fisiología , Células Cultivadas , Fosfatidilinositol 3-Quinasas Clase II/genética , Modelos Animales de Enfermedad , Isoenzimas , Ratones , Ratones Noqueados , Contracción Muscular/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Proteínas de Unión al GTP rho/genéticaRESUMEN
Autophagy is subdivided into chaperone-mediated autophagy, microautophagy and macroautophagy and is a highly conserved intracellular degradative pathway. It is crucial for cellular homeostasis and also serves as a response to different stresses. Here we focus on macroautophagy, which targets damaged organelles and large protein assemblies, as well as pathogenic intracellular microbes for destruction. During this process, cytosolic material becomes enclosed in newly generated double-membrane vesicles, the so-called autophagosomes. Upon maturation, the autophagosome fuses with the lysosome for degradation of the cargo. The basic molecular machinery that controls macroautophagy works in a sequential order and consists of the ATG1 complex, the PtdIns3K complex, the membrane delivery system, two ubiquitin-like conjugation systems, and autophagy adaptors and receptors. Since the different stages of macroautophagy from initiation to final degradation of cargo are tightly regulated and highly conserved across eukaryotes, simple model organisms in combination with a wide range of techniques contributed significantly to advance our understanding of this complex dynamic process. Here, we present the social amoeba Dictyostelium discoideum as an advantageous and relevant experimental model system for the analysis of macroautophagy.
Asunto(s)
Autofagosomas , Autofagia , Dictyostelium/fisiología , Animales , Caenorhabditis elegans , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Citosol/metabolismo , Dictyostelium/genética , Drosophila melanogaster , Homeostasis , Lisosomas/metabolismo , Fagocitosis , Saccharomyces cerevisiae , Ubiquitina/metabolismoRESUMEN
BACKGROUND: Alteration of signalling pathways regulating cell cycle progression is a common feature of cancer cells. Several drugs targeting distinct phases of the cell cycle have been developed but the inability of many of them to discriminate between normal and cancer cells has strongly limited their clinical potential because of their reduced efficacy at the concentrations used to limit adverse side effects. Mechanisms of resistance have also been described, further affecting their efficacy. Identification of novel targets that can potentiate the effect of these drugs or overcome drug resistance can provide a useful strategy to exploit the anti-cancer properties of these agents to their fullest. METHODS: The class II PI3K isoform PI3K-C2ß was downregulated in prostate cancer PC3 cells and cervical cancer HeLa cells using selective siRNAs and the effect on cell growth was determined in the absence or presence of the microtubule-stabilizing agent/anti-cancer drug docetaxel. Mitosis progression was monitored by time-lapse microscopy. Clonogenic assays were performed to determine the ability of PC3 and HeLa cells to form colonies upon PI3K-C2ß downregulation in the absence or presence of docetaxel. Cell multi-nucleation was assessed by immunofluorescence. Tumour growth in vivo was assessed using a xenograft model of PC3 cells upon PI3K-C2ß downregulation and in combination with docetaxel. RESULTS: Downregulation of PI3K-C2ß delays mitosis progression in PC3 and HeLa cells, resulting in reduced ability to form colonies in clonogenic assays in vitro. Compared to control cells, PC3 cells lacking PI3K-C2ß form smaller and more compact colonies in vitro and they form tumours more slowly in vivo in the first weeks after cells implant. Stable and transient PI3K-C2ß downregulation potentiates the effect of low concentrations of docetaxel on cancer cell growth. Combination of PI3K-C2ß downregulation and docetaxel almost completely prevents colonies formation in clonogenic assays in vitro and strongly inhibits tumour growth in vivo. CONCLUSIONS: These data reveal a novel role for the class II PI3K PI3K-C2ß during mitosis progression. Furthermore, data indicate that blockade of PI3K-C2ß might represent a novel strategy to potentiate the effect of docetaxel on cancer cell growth.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Docetaxel/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/enzimología , Animales , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Femenino , Células HeLa , Humanos , Masculino , Ratones Desnudos , Células PC-3 , Neoplasias de la Próstata/patología , Distribución Aleatoria , Transfección , Neoplasias del Cuello Uterino/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide because of rapid progression and high incidence of metastasis or recurrence. Accumulating evidence shows that CD73-expressing tumor cell is implicated in development of several types of cancer. However, the role of CD73 in HCC cell has not been systematically investigated and its underlying mechanism remains elusive. METHODS: CD73 expression in HCC cell was determined by RT-PCR, Western blot, and immunohistochemistry staining. Clinical significance of CD73 was evaluated by Cox regression analysis. Cell counting kit-8 and colony formation assays were used for proliferation evaluation. Transwell assays were used for motility evaluations. Co-immunoprecipitation, cytosolic and plasma membrane fractionation separation, and ELISA were applied for evaluating membrane localization of P110ß and its catalytic activity. NOD/SCID/γc(null) (NOG) mice model was used to investigate the in vivo functions of CD73. RESULTS: In the present study, we demonstrate that CD73 was crucial for epithelial-mesenchymal transition (EMT), progression and metastasis in HCC. CD73 expression is increased in HCC cells and correlated with aggressive clinicopathological characteristics. Clinically, CD73 is identified as an independent poor prognostic indicator for both time to recurrence and overall survival. CD73 knockdown dramatically inhibits HCC cells proliferation, migration, invasion, and EMT in vitro and hinders tumor growth and metastasis in vivo. Opposite results could be observed when CD73 is overexpressed. Mechanistically, adenosine produced by CD73 binds to adenosine A2A receptor (A2AR) and activates Rap1, which recruits P110ß to the plasma membrane and triggers PIP3 production, thereby promoting AKT phosphorylation in HCC cells. Notably, a combination of anti-CD73 and anti-A2AR achieves synergistic depression effects on HCC growth and metastasis than single agent alone. CONCLUSIONS: CD73 promotes progression and metastasis through activating PI3K/AKT signaling, indicating a novel prognostic biomarker for HCC. Our data demonstrate the importance of CD73 in HCC in addition to its immunosuppressive functions and revealed that co-targeting CD73 and A2AR strategy may be a promising novel therapeutic strategy for future HCC management.
Asunto(s)
5'-Nucleotidasa/metabolismo , Carcinoma Hepatocelular/metabolismo , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión a Telómeros/metabolismo , 5'-Nucleotidasa/genética , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasas Clase II/genética , Progresión de la Enfermedad , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Xenoinjertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/metabolismo , Complejo Shelterina , Transducción de Señal , Proteínas de Unión a Telómeros/genéticaRESUMEN
The phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylate inositol phospholipids, thereby controlling membrane lipid composition and regulating a wide range of intracellular processes, including vesicular trafficking and signal transduction. Despite the vast knowledge on class I PI3Ks, recent studies are only now revealing the importance of class II PI3Ks in cell proliferation, survival, and migration. Increasing evidence suggests that the three class II PI3Ks isoforms (PI3K-C2α, PI3K-C2ß, and PI3K-C2γ) have distinct and non-overlapping cellular roles. Here, we focus on the cellular functions of class II PI3Ks in different cell systems and underline the emerging importance of these enzymes in various physiological and pathological contexts.
Asunto(s)
Células/citología , Células/enzimología , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Enfermedades Metabólicas/enzimología , Neoplasias/enzimología , Animales , Células/patología , Humanos , Enfermedades Metabólicas/patología , Neoplasias/patología , Transducción de SeñalRESUMEN
Class II phosphoinositide 3-kinases (PI3Ks), PI3K-C2α and PI3K-C2ß, are highly homologous and distinct from class I and class III PI3Ks in catalytic products and domain structures. In contrast to class I and class III PI3Ks, physiological roles of PI3K-C2α and PI3K-C2ß are not fully understood. Because we previously demonstrated that PI3K-C2α is involved in vascular smooth muscle contraction, we studied the phenotypes of smooth muscle-specific knockout (KO) mice of PI3K-C2α and PI3K-C2ß. The pup numbers born from single PI3K-C2α-KO and single PI3K-C2ß-KO mothers were similar to those of control mothers, but those from double KO (DKO) mothers were smaller compared with control mice. However, the number of intrauterine fetuses in pregnant DKO mothers was similar to that in control mice. Both spontaneous and oxytocin-induced contraction of isolated uterine smooth muscle (USM) strips was diminished in DKO mice but not in either of the single KO mice, compared with control mice. Furthermore, contraction of USM of DKO mice was less sensitive to a Rho kinase inhibitor. Mechanistically, the extent of oxytocin-induced myosin light chain phosphorylation was greatly reduced in USM from DKO mice compared with control mice. The oxytocin-induced rise in the intracellular Ca2+ concentration in USM was similar in DKO and control mice. However, Rho activation in the intracellular compartment was substantially attenuated in DKO mice compared with control mice, as evaluated by fluorescence resonance energy transfer imaging technique. These data indicate that both PI3K-C2α and PI3K-C2ß are required for normal USM contraction and parturition mainly through their involvement in Rho activation.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Músculo Liso Vascular/enzimología , Parto , Fosfatidilinositol 3-Quinasas/metabolismo , Contracción Uterina , Útero/enzimología , Proteína de Unión al GTP rhoA/metabolismo , Animales , Fosfatidilinositol 3-Quinasas Clase II/genética , Femenino , Ratones , Ratones Noqueados , Contracción Muscular , Músculo Liso Vascular/fisiología , Cadenas Ligeras de Miosina , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Útero/fisiología , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Pinocytosis is an important fundamental cellular process that is used by the cell to transport fluid and solutes. Phosphoinositide 3-kinases (PI3Ks) regulate a diverse array of dynamic membrane events. However, it is not well-understood which PI3K isoforms are involved in specific mechanisms of pinocytosis. We performed knockdown studies of endogenous PI3K isoforms and clathrin heavy chain (CHC) mediated by small interfering RNA (siRNA). The results demonstrated that the class II PI3K PI3K-C2α and PI3K-C2ß, but not the class I or III PI3K, were required for pinocytosis, based on an evaluation of fluorescein-5-isothiocyanate (FITC)-dextran uptake in endothelial cells. Pinocytosis was partially dependent on both clathrin and dynamin, and both PI3K-C2α and PI3K-C2ß were required for clathrin-mediated-but not clathrin-non-mediated-FITC-dextran uptake at the step leading up to its delivery to early endosomes. Both PI3K-C2α and PI3K-C2ß were co-localized with clathrin-coated pits and vesicles. However, PI3K-C2ß, but not PI3K-C2α, was highly co-localized with actin filament-associated clathrin-coated structures and required for actin filament formation at the clathrin-coated structures. These results indicate that PI3K-C2α and PI3K-C2ß play differential, indispensable roles in clathrin-mediated pinocytosis.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Clatrina/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Pinocitosis/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana , Humanos , ARN Interferente Pequeño/metabolismoRESUMEN
X-linked myotubular myopathy (XLMTM, also known as XLCNM) is a severe congenital muscular disorder due to mutations in the myotubularin gene, MTM1. It is characterized by generalized hypotonia, leading to neonatal death of most patients. No specific treatment exists. Here, we show that tamoxifen, a well-known drug used against breast cancer, rescues the phenotype of Mtm1-deficient mice. Tamoxifen increases lifespan several-fold while improving overall motor function and preventing disease progression including lower limb paralysis. Tamoxifen corrects functional, histological and molecular hallmarks of XLMTM, with improved force output, myonuclei positioning, myofibrillar structure, triad number, and excitation-contraction coupling. Tamoxifen normalizes the expression level of the XLMTM disease modifiers DNM2 and PI3KC2B, likely contributing to the phenotypic rescue. Our findings demonstrate that tamoxifen is a promising candidate for clinical evaluation in XLMTM patients.
Asunto(s)
Actividad Motora/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Miopatías Estructurales Congénitas/tratamiento farmacológico , Sustancias Protectoras/farmacología , Proteínas Tirosina Fosfatasas no Receptoras/genética , Tamoxifeno/farmacología , Animales , Fosfatidilinositol 3-Quinasas Clase II/genética , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Dinamina II/genética , Dinamina II/metabolismo , Estimulación Eléctrica , Acoplamiento Excitación-Contracción/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Genes Letales , Humanos , Longevidad/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Miofibrillas/efectos de los fármacos , Miofibrillas/metabolismo , Miofibrillas/ultraestructura , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/metabolismo , Miopatías Estructurales Congénitas/patología , Proteínas Tirosina Fosfatasas no Receptoras/deficienciaRESUMEN
Class II phosphoinositide 3-kinases (PI3K-C2) are large multidomain enzymes that control cellular functions ranging from membrane dynamics to cell signaling via synthesis of 3'-phosphorylated phosphoinositides. Activity of the alpha isoform (PI3K-C2α) is associated with endocytosis, angiogenesis, and glucose metabolism. How PI3K-C2α activity is controlled at sites of endocytosis remains largely enigmatic. Here we show that the lipid-binding PX-C2 module unique to class II PI3Ks autoinhibits kinase activity in solution but is essential for full enzymatic activity at PtdIns(4,5)P2-rich membranes. Using HDX-MS, we show that the PX-C2 module folds back onto the kinase domain, inhibiting its basal activity. Destabilization of this intramolecular contact increases PI3K-C2α activity in vitro and in cells, leading to accumulation of its lipid product, increased recruitment of the endocytic effector SNX9, and facilitated endocytosis. Our studies uncover a regulatory mechanism in which coincident binding of phosphoinositide substrate and cofactor selectively activate PI3K-C2α at sites of endocytosis.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Fosfatidilinositol 3-Quinasas Clase II/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Animales , Dominios C2/fisiología , Células COS , Chlorocebus aethiops , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/fisiología , Clatrina/fisiología , Endocitosis/fisiología , Células HEK293 , Homeostasis , Humanos , Lípidos/fisiología , Espectrometría de Masas , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Unión Proteica , Dominios Proteicos , Transducción de SeñalRESUMEN
Bornyl cis-4-hydroxycinnamate, a bioactive compound isolated from Piper betle stems, has the potential for use as an anti-cancer agent. This study investigated the effects of bornyl cis-4-hydroxycinnamate on cell migration and invasion in melanoma cells. Cell migration and invasion were compared in A2058 and A375 melanoma cell lines treated with/without bornyl cis-4-hydroxycinnamate (1â»6 µM). To examine whether bornyl cis-4-hydroxycinnamate has a potential anti-metastatic effect on melanoma cells, cell migration and invasion assays were performed using a Boyden chamber assay and a transwell chamber in A2058 and A375 cells. Gelatin zymography was employed to determine the enzyme activities of MMP-2 and MMP-9. Cell lysates were collected for Western blotting analysis of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors of metalloproteinase-1/2 (TIMP-1/2), as well as key molecules in the mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK)/ phosphatidylinositide-3 kinases (PI3K)/Akt/ mammalian target of rapamycin (mTOR), growth factor receptor-bound protein 2 (GRB2) signaling pathways. Our results demonstrated that bornyl cis-4-hydroxycinnamate is a potentially useful agent that inhibits melanoma cell migration and invasion, and altered melanoma cell metastasis by reducing MMP-2 and MMP-9 expression through inhibition of the FAK/PI3K/Akt/mTOR, MAPK, and GRB2 signaling pathways. Moreover, bornyl cis-4-hydroxycinnamate inhibited the process of the epithelial-to-mesenchymal transition in A2058 and A375 melanoma cells. These findings suggested that bornyl cis-4-hydroxycinnamate has potential as a chemotherapeutic agent, and warrants further investigation for its use in the management of human melanoma.
Asunto(s)
Antineoplásicos/farmacología , Canfanos/farmacología , Ácidos Cumáricos/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/tratamiento farmacológico , Fitoquímicos/farmacología , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Relación Dosis-Respuesta a Droga , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Hidróxidos/química , Invasividad Neoplásica , Metástasis de la Neoplasia , Fitoquímicos/química , Fitoquímicos/aislamiento & purificación , Fitoquímicos/uso terapéutico , Piper betle/química , Tallos de la Planta/química , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Presynaptic homeostatic plasticity stabilizes information transfer at synaptic connections in organisms ranging from insect to human. By analogy with principles of engineering and control theory, the molecular implementation of PHP is thought to require postsynaptic signaling modules that encode homeostatic sensors, a set point, and a controller that regulates transsynaptic negative feedback. The molecular basis for these postsynaptic, homeostatic signaling elements remains unknown. Here, an electrophysiology-based screen of the Drosophila kinome and phosphatome defines a postsynaptic signaling platform that includes a required function for PI3K-cII, PI3K-cIII and the small GTPase Rab11 during the rapid and sustained expression of PHP. We present evidence that PI3K-cII localizes to Golgi-derived, clathrin-positive vesicles and is necessary to generate an endosomal pool of PI(3)P that recruits Rab11 to recycling endosomal membranes. A morphologically distinct subdivision of this platform concentrates postsynaptically where we propose it functions as a homeostatic controller for retrograde, trans-synaptic signaling.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Plasticidad Neuronal , Terminales Presinápticos/fisiología , Transducción de Señal , Animales , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Fenómenos Electrofisiológicos , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Nutrient sensing by mechanistic target of rapamycin complex 1 (mTORC1) on lysosomes and late endosomes (LyLEs) regulates cell growth. Many factors stimulate mTORC1 activity, including the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] by class I phosphatidylinositol 3-kinases (PI3Ks) at the plasma membrane. We investigated mechanisms that repress mTORC1 under conditions of growth factor deprivation. We identified phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], synthesized by class II PI3K ß (PI3KC2ß) at LyLEs, as a negative regulator of mTORC1, whereas loss of PI3KC2ß hyperactivated mTORC1. Growth factor deprivation induced the association of PI3KC2ß with the Raptor subunit of mTORC1. Local PI(3,4)P2 synthesis triggered repression of mTORC1 activity through association of Raptor with inhibitory 14-3-3 proteins. These results unravel an unexpected function for local PI(3,4)P2 production in shutting off mTORC1.
Asunto(s)
Endosomas/enzimología , Lisosomas/enzimología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Fosfatidilinositol 3-Quinasas Clase II/genética , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Activación Enzimática/fisiología , Fibroblastos , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Transporte de Proteínas/genética , Proteína Reguladora Asociada a mTOR/metabolismo , Transducción de Señal/genéticaRESUMEN
A key component during sepsis is the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, of which the PI3K-γ isoform is a major regulator in many inflammatory responses. However, the role of PI3K-γ in the development of sepsis-induced myocardial dysfunction (SIMD) is unknown. In this study, we established a model of SIMD induced by lipopolysaccharide (LPS), subsequently used the selective inhibitor LY294002 and AS605240 to block the effect of PI3K and PI3K-γ, respectively. Cardiac function was evaluated by echocardiography, hearts were obtained for histological and protein expression examinations. ELISA was used to measure the serum levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), cardiac troponin I (cTnI) and heart-type fatty acid binding protein (H-FABP). LPS-treated mice showed an increase to cardiac inflammation, myocardial damage and production of TNF-α, IL-6, NF-κB, cTnI and H-FABP. Administration of AS605240 to LPS-treated mice reduced some patho-physiological characteristics of SIMD and reduced TNF-α, IL-6, cTnI and H-FABP production. However, administration of LY294002 did not improve those same conditions. The results showed that PI3K-γ is likely a crucial element in SIMD by regulating the PI3K/Akt pathway, and become a new marker of myocardial injury. Inhibition of PI3K-γ might be a potential therapeutic target in SIMD.
Asunto(s)
Cardiomiopatías/metabolismo , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Sepsis/complicaciones , Animales , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Cromonas/administración & dosificación , Fosfatidilinositol 3-Quinasas Clase II/genética , Citocinas/sangre , Modelos Animales de Enfermedad , Regulación hacia Abajo , Interleucina-6/biosíntesis , Lipopolisacáridos/administración & dosificación , Ratones , Morfolinas/administración & dosificación , Miocardio/patología , Quinoxalinas/administración & dosificación , Quinoxalinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Tiazolidinedionas/administración & dosificación , Tiazolidinedionas/uso terapéutico , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Phosphatidylinositol-3-phosphate (PI3P) is a key player in membrane dynamics and trafficking regulation. Most PI3P is associated with endosomal membranes and with the autophagosome preassembly machinery, presumably at the endoplasmic reticulum. The enzyme responsible for most PI3P synthesis, VPS34 and proteins such as Beclin1 and ATG14L that regulate PI3P levels are positive modulators of autophagy initiation. It had been assumed that a local PI3P pool was present at autophagosomes and preautophagosomal structures, such as the omegasome and the phagophore. This was recently confirmed by the demonstration that PI3P-binding proteins participate in the complex sequence of signalling that results in autophagosome assembly and activity. Here we summarize the historical discoveries of PI3P lipid kinase involvement in autophagy, and we discuss the proposed role of PI3P during autophagy, notably during the autophagosome biogenesis sequence.
Asunto(s)
Autofagosomas/fisiología , Autofagia , Microdominios de Membrana/fisiología , Modelos Biológicos , Biogénesis de Organelos , Fosfatos de Fosfatidilinositol/metabolismo , Sistemas de Mensajero Secundario , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Autofagosomas/enzimología , Proteínas Relacionadas con la Autofagia/metabolismo , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/fisiología , Endosomas/enzimología , Endosomas/fisiología , Humanos , Lisosomas/enzimología , Lisosomas/fisiología , Microdominios de Membrana/enzimologíaRESUMEN
BACKGROUND: Class II phosphoinositide 3-kinase α-isoform (PI3K-C2α) is involved in regulating KCl-induced vascular smooth muscle contraction. The current study was to investigate the effects of sevoflurane (SEVO) and isoflurane (ISO) on KCl-elicited PI3KC2α mediated vasoconstriction in rat aortic smooth muscle. METHODS: Isometric force, in the absence or presence of SEVO or ISO (1 ~ 3 minimum alveolar concentration, MAC), PI3K inhibitor LY294002, Rho kinase inhibitor Y27632, and membrane translocation of PI3K-p85, PI3K-C2α, Rho kinase (Rock II), or phosphorylation of MYPT1/Thr853, MYPT1/Thr696, CPI-17/Thr38 and MLC in response to KCl (60 mM) was measured by using isometric force transducer and western blotting analysis, respectively. RESULTS: KCl elicited a rapid and sustained contraction of rat aortic smooth muscle that was inhibited by both SEVO and ISO in a concentration-dependent manner, and also suppressed by LY294002 (1 mM) and Y27632 (1 uM). LY294002 (1 mM) and Y27632 (1 uM) also inhibited KCl-induced MLC phosphorylation. LY294002 (1 mM) inhibited KCl-induced PI3K-p85, PI3K-C2α membrane translocation in response to KCl (p <0.05, p < 0.01, respectively). Not only Y27632 (1 uM), but also LY294002 (1 mM), inhibited KCl-induced Rock-II membrane translocation (p < 0.01). SEVO and ISO inhibited KCl-stimulated MLC phosphorylation, PI3K-C2α and Rock-II,not PI3K p85 membrane translocation in a concentration-dependent manner in rat aorta. Both SEVO and ISO suppressed the MYPT1/Thr853, not MYPT1/Thr696 and CPI-17/Thr38, MLC phosphorylation in response to KCl. CONCLUSION: PI3K-C2α mediates part of SEVO and ISO-mediated vasodilation in rat aorta. The cellular mechanisms underlying the inhibitory effect of volatile anesthetics might be mediated by KCl/PI3K-C2α/Rho kinase/MYPT1/MLC pathway.