RESUMEN
Pten controls a signaling axis that is implicated to regulate cell proliferation, growth, survival, migration, and metabolism. The molecular mechanisms underlying the specificity of Pten responses to such diverse cellular functions are currently poorly understood. Here we report the control of Pten activity and signaling specificity during the cell cycle by Ndfip1 regulation of Pten spatial distribution. Genetic deletion of Ndfip1 resulted in a loss of Pten nuclear compartmentalization and increased cell proliferation, despite cytoplasmic Pten remaining active in regulating PI3K/Akt signaling. Cells lacking nuclear Pten were found to have dysregulated levels of Plk1 and cyclin D1 that could drive cell proliferation. In vivo, transgene expression of Ndfip1 in the developing brain increased nuclear Pten and lengthened the cell cycle of neuronal progenitors, resulting in microencephaly. Our results show that local partitioning of Pten from the cytoplasm to the nucleus represents a key mechanism contributing to the specificity of Pten signaling during cell proliferation.
Asunto(s)
Proteínas Portadoras/fisiología , Proliferación Celular , Proteínas de la Membrana/fisiología , Fosfohidrolasa PTEN/metabolismo , Transporte Activo de Núcleo Celular , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Ciclina D1/metabolismo , Femenino , Indazoles/farmacología , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Microcefalia/metabolismo , Células PC12 , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Transducción de Señal , Sirolimus/farmacología , Sulfonamidas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Quinasa Tipo Polo 1RESUMEN
During injury, cells are vulnerable to apoptosis from a variety of stress conditions including DNA damage causing double-stranded breaks. Without repair, these breaks lead to aberrations in DNA replication and transcription, leading to apoptosis. A major response to DNA damage is provided by the protein kinase ATM (ataxia telangiectasia mutated) that is capable of commanding a plethora of signaling networks for DNA repair, cell cycle arrest, and even apoptosis. A key element in the DNA damage response is the mobilization of activating proteins into the cell nucleus to repair damaged DNA. BRAT1 is one of these proteins, and it functions as an activator of ATM by maintaining its phosphorylated status while also keeping other phosphatases at bay. However, it is unknown how BRAT1 is trafficked into the cell nucleus to maintain ATM phosphorylation. Here we demonstrate that Ndfip1-mediated ubiquitination of BRAT1 leads to BRAT1 trafficking into the cell nucleus. Without Ndfip1, BRAT1 failed to translocate to the nucleus. Under genotoxic stress, cells showed increased expression of both Ndfip1 and phosphorylated ATM. Following brain injury, neurons show increased expression of Ndfip1 and nuclear translocation of BRAT1. These results point to Ndfip1 as a sensor protein during cell injury and Ndfip1 up-regulation as a cue for BRAT1 ubiquitination by Nedd4 E3 ligases, followed by nuclear translocation of BRAT1.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Portadoras/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Lesiones Encefálicas/metabolismo , Línea Celular , Daño del ADN , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones Endogámicos C57BL , Ubiquitina-Proteína Ligasas Nedd4 , Unión Proteica , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteolisis , Transducción de Señal , UbiquitinaciónRESUMEN
PTEN was discovered as a membrane-associated tumor suppressor protein nearly two decades ago, but the concept that it can be secreted and taken up by recipient cells is revolutionary. Since then, various laboratories have reported that PTEN is indeed secreted and available for uptake by other cells in at least two different guises. First, PTEN may be packaged and exported within extracellular vesicles (EV) called exosomes. Second, PTEN may also be secreted as a naked protein in a longer isoform called PTEN-long. While the conditions favouring the secretion of PTEN-long remain unknown, PTEN secretion in exosomes is enhanced by the Ndfip1/Nedd4 ubiquitination system. In this report, we describe conditions for packaging PTEN in exosomes and their potential use for mediating non cell-autonomous functions in recipient cells. We suggest that this mode of PTEN transfer may potentially provide beneficial PTEN for tumor suppression, however it may also propagate deleterious versions of mutated PTEN causing tumorigenesis.
Asunto(s)
Exosomas/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , RatonesRESUMEN
The spatial regulation of Pten is critical for its role as a tumour suppressor with both nuclear and cytoplasmic locations being implicated with distinct functions. In the cytoplasm, Pten plays a central role in opposing PI3K/Akt cell signalling, whereas in the nucleus, Pten is important for maintaining genome stability and enhancing the tumour suppressor activity of APC-CDH1. Despite this diversity in protein function at different subcellular locations, there is limited knowledge on how Pten is able to find different cellular niches. Here, we report that Rab5 GTPase is required for efficient trafficking and ubiquitination of Pten on endosomes inside the cytosol. Using bimolecular fluorescence complementation (BiFC) for imaging protein interactions, we observed that ubiquitinated Pten is localized to peri-nuclear and nuclear regions of the cell. Nuclear trafficking of Pten required both Rab5 as well as the E3 ligase adaptor protein Ndfip1. Rab5 colocalization with Pten was observed on endosomes and expression of a dominant negative form of Rab5 significantly reduced Pten ubiquitination and nuclear trafficking. Genomic deletion of Ndfip1 abrogated nuclear trafficking of ubiquitinated Pten, even in the presence of Rab5. Our findings show that endosomal trafficking and ubiquitination are important mechanisms for the subcellular distribution of Pten.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Animales , Células Cultivadas , Endosomas/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Ratones , Transporte de Proteínas , UbiquitinaciónRESUMEN
Ubiquitin ligases of the Nedd4 family are important for axon and dendrite development, but little is known about their adaptor, Nedd4 family-interacting protein 1 (Ndfip1), that is responsible for their enzymatic activation. To study the function of Ndfip1 in cortical development, we generated a conditional knock-out (conditional KO) in neurons. The Ndfip1 conditional KO mice were viable; however, cortical neurons in the adult brain exhibited atrophic characteristics, including stunted dendritic arbors, blebbing of dendrites, and fewer dendritic spines. In electron micrographs, these neurons appeared shrunken with compacted somata and involutions of the nuclear membrane. In culture, Ndfip1 KO neurons exhibited exuberant sprouting suggesting loss of developmental control. Biochemical analysis of postsynaptic density (PSD) fractions from Ndfip1 KO cortical and hippocampal neurons showed that the postsynaptic proteins (Arc and PSD-95) were reduced compared with wild-type controls. In addition, the PI3 kinase/Akt signaling pathway was altered. These results indicate that Ndfip1, through its Nedd4 effectors, is important for the development of dendrites and dendritic spines in the cortex.
Asunto(s)
Proteínas Portadoras/genética , Espinas Dendríticas/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de la Membrana/genética , Neocórtex , Células Piramidales/diagnóstico por imagen , Animales , Animales Recién Nacidos , Fraccionamiento Celular , Células Cultivadas , Homólogo 4 de la Proteína Discs Large , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Guanilato-Quinasas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neocórtex/citología , Neocórtex/embriología , Neocórtex/crecimiento & desarrollo , Nestina/genética , Nestina/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , UltrasonografíaRESUMEN
There is controversy whether accumulation of the tumor suppressor PTEN protein in the cell nucleus under stress conditions such as trauma and stroke causes cell death. A number of in vitro studies have reported enhanced apoptosis in neurons possessing nuclear PTEN, with the interpretation that its nuclear phosphatase activity leads to reduction of the survival protein phospho-Akt. However, there have been no in vivo studies to show that nuclear PTEN in neurons under stress is detrimental. Using a mouse model of injury, we demonstrate here that brain trauma altered the nucleo-cytoplasmic distribution of Pten, resulting in increased nuclear Pten but only in surviving neurons near the lesion. This event was driven by Ndfip1, an adaptor and activator of protein ubiquitination by Nedd4 E3 ligases. Neurons next to the lesion with nuclear PTEN were invariably negative for TUNEL, a marker for cell death. These neurons also showed increased Ndfip1 which we previously showed to be associated with neuron survival. Biochemical assays revealed that overall levels of Pten in the affected cortex were unchanged after trauma, suggesting that Pten abundance globally had not increased but rather Pten subcellular location in affected neurons had changed. Following experimental injury, the number of neurons with nuclear Pten was reduced in heterozygous mice (Ndfip1(+/-)) although lesion volumes were increased. We conclude that nuclear trafficking of Pten following injury leads to neuron survival not death.
Asunto(s)
Lesiones Encefálicas/patología , Núcleo Celular/metabolismo , Regulación de la Expresión Génica/fisiología , Neuronas , Fosfohidrolasa PTEN/metabolismo , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/fisiología , Proteínas Portadoras/genética , Supervivencia Celular/genética , Citoplasma , Modelos Animales de Enfermedad , Lateralidad Funcional , Inmunoprecipitación , Etiquetado Corte-Fin in Situ , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Proteína Oncogénica v-akt , Fosfohidrolasa PTEN/genética , Transporte de Proteínas/genéticaRESUMEN
During development, protein substrates need to be removed and degraded when they are no longer required. The E3 ubiquitin ligases, including Nedd4 family proteins, are a major group of enzymes responsible for adding ubiquitin chains to protein substrates prior to their degradation. Ndfip1 (Nedd4 family-interacting protein 1) is an adaptor and activator for Nedd4-family ubiquitin ligases for increasing substrate specificity. To study Nedd4-mediated ubiquitination during cortical development, we have mapped the spatio-temporal dynamics of Ndfip1 protein expression by immunocytochemistry. Ndfip1 expression was observed from embryonic day 11 (E11.5) until adult stages. Its presence increased during the postnatal stages and peaked at postnatal day 7 (P7). Spatially, Ndfip1 was found in the ventricular and marginal zones during corticogenesis but also in the cortical plate and subplate during midstage cortical development (E15.5). Postnatally, Ndfip1 was expressed in all cortical neurons (but not in glial cells) and this expression was both ubiquitous and uniform across cortical layers involving both pyramidal and non-pyramidal neurons. This consistent but dynamic pattern of Ndfip1 expression in temporal and spatial domains of the cortical landscape is indicative of complex programs of protein ubiquitination during corticogenesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Corteza Cerebral/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Interneuronas/metabolismo , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Ubiquitina-Proteína Ligasas Nedd4 , Células Piramidales/metabolismo , UbiquitinaciónRESUMEN
Exosomes are microvesicles of endosomal origin that are secreted, and their contents (proteins, lipids, DNA, or microRNAs) can alter the physiological states of recipient cells. We demonstrated that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein normally localized in the cytoplasm and nucleus, was secreted in exosomes. Secreted PTEN was internalized by recipient cells with resultant functional activity, which resulted in reduced phosphorylation of the serine and threonine kinase Akt and reduced cellular proliferation. PTEN secretion in exosomes required Ndfip1, an adaptor protein for members of the Nedd4 family of E3 ubiquitin ligases. Without Ndfip1, neither Nedd4-1 nor Nedd4-2 promoted the recruitment of PTEN into exosomes. In addition, lysine 13 within PTEN, which is required for its ubiquitination by Nedd4-1, was required for exosomal transport of PTEN. These results implicate Ndfip1 as a molecular regulator of the exosomal export of PTEN, with consequences for non-cell-autonomous PTEN activity. Thus, we suggest that the ability of PTEN to exert phosphatase activity beyond the cell in which it is produced has implications for PTEN function during development, health, and disease.
Asunto(s)
Exosomas/metabolismo , Proteína Oncogénica v-akt/metabolismo , Fosfohidrolasa PTEN/metabolismo , Animales , Western Blotting , Proteínas Portadoras/metabolismo , Proliferación Celular , Electroforesis en Gel de Poliacrilamida , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica , Ubiquitina-Proteína Ligasas Nedd4 , Fosforilación , Sales de Tetrazolio , Tiazoles , Azul de Tripano , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
PTEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regulator of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiological events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was translocated into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family-interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overexpression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1(-/-) fibroblasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival.